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The function of the structure of DNA in chromosomes
BIOCHIMIE, 1972, 54, 1005-1011.
The ihnction of the structure of DNA in chromosomes. S t a n l e y BRAI~I.
D~partement de Biologie Mol~culaire, lnstitut Pasteur, Paris 15, France. (3/6/1972).
Summary. - - The proposed model can be summarized as follows : 1) Most of the DNA in chromosomes is non-uniformly folded into a compact left handed super coil whose average pitch and radius are 45 A. The pitch or radius of this double helical DNA are 10 p. cent smaller than in solution. 2) By virtue of its compact secondary structure this m a j o r fraction of the D NA cannot be transcribed ; concurrently, the compact tertiary structure blocks the access of recognition ~and polymerase molecules to this DNA. 3) A smal:l fraction of the total DNA is not tightly folded and is thereby marked for recognition. It is suggested that this recognition DNA is of non-typical base composition and that it is very rich in AT. The recognition DNA is kept unfolded by its unique secondary structure and by preferential interaction with lysine rich histories. 4) Specific recognition may involve the less specific binding to a general type of DNA (very AT rich DNA) followed by a recognizer-induced transition to allow for productive binding to the proper sequence. 5) It is proposed thai in the cell very AT rich DNA cannot initiate transcription due to its unique secondary structure and because it does not adopt the A-form.
INTRODUCTION. A m o d e l is p r o p o s e d h e r e for t h e s t r u c t u r e a n d f u n c t i o n of D N A in c h r o m o s o m e s of h i g h e r o r g a n i s m s . T h e m o d e l is b a s e d u p o n s t r u c t u r a l s t u d i e s of n u c l e o h i s t o n e a n d D NA. It w i l l p r o v i d e n e i t h e r a c o m p l e t e n o r e x a c t d e s c r i p t i o n of t h e c h r o m o some. My p u r p o s e is to a t t e m p t to g i v e a f r a m e of r e f e r e n c e w h i c h w i l l a i d in t h e r e l a t i n g of s t r u c t u r a l s t u d i e s of c h r o m o s o i n e s a n d n u c l e i c a c i d s "to t h e f u n c t i o n of DNA. T h e f u n c t i o n of t h e s t r u c t u r e of t h e c h r o m o s o m e is to e f f i c i e n t l y h a n d l e l o n g D N A m o l e c u l e s . It p r o v i d e s for o r d e r e d t r a n s c r i p t i o n , r e p l i c a t i o n a n d m i t o t i c s e g r e g a t i o n . T h e c h r o m o s o m e c o n s i s t s of D,NA i n t i m a t e l y a s s o c i a t e d w i t h a b a s i c p r o t e i n - h i s t o n e - - a n d v a r i a b l e a m o u n t s of n o n h i s t o n e a c i d p r o t e i n a n d RNA. T h e D N A - h i s t o n e c o m p l e x w h i c h w e call n u c l e o h i s t o n e is r e p r e s s e d w i t h resp e c t to p r o t e i n s y n t h e s i s . T h a t is, it m a k e s v e r y little or no m - R N A w i t h R N A p o l y m e r a s e in vitro, a n d it is t h e d o m i n a n t f o r m of c h r o m a t i n in m e t a b o l i c a l l y i n a c t i v e cells. N u c l e o h i s t o n e is o r g a n i z e d i n t o fibers t h a t fold o r coil up to m a k e t h e c o n d e n sed c h r o m o s o m e . C h r o m o s o m e s in t h e c o n d e n s e d state h a v e a d i a m e t e r on t h e o r d e r of 2 00010 000 A a n d e l e c t r o n m i c r o s c o p i s t s [1, 2] h a v e s h o w n t h a t t h e y c o n s i s t of fibers 200-250 A in diam e t e r . Ris has s h o w n t h a t if t h e 20'0 A fibrils a r e t r e a t e d w i t h c h e l a t i n g agents to r e m o v e c a l c i u m t h e y a r e g r a d u a l l y t r a n s f o r m e d i n t o fibers 80-100 A in d i a m e t e r [3]. He suggests t h a t t h e 200 A fiber
c o n s i s t s of 100 A fibers f o l d e d b a c k u p o n t h e m s e l v e s or a g g r e g a t e d to m a k e t h e t h i c k e r fiber. It is t h e 100 A n u c l e o h i s t o n e fiber w h i c h m o s t w o r k e r s c o n s i d e r to be t h e b a s i c c h r o m o s o m a l u n i t a n d h e r e I w i s h to r e l a t e its t e r t i a r y , s e c o n d a r y , a n d p r i m a r y s t r u c t u r e to t h e c o n t r o l of gene a c t i v i t y . T w o d e t a i l e d s t r u c t u r a l m o d e l s for n u c l e o h i s t o n e h a v e b e e n p r o p o s e d - t h a t of P a r d o n , W i l k i n s a n d R i c h a r d s [4] a n d t h a t of B r a m a n d Ris. [5, 6] (see f i g u r e 1). T h e f o r m e r is a l o o s e s u p e r c o i l of p i t c h 120 A a n d o u t e r d i a m e t e r 130 A. T h e o t h e r is a c o m p a c t n o n - u n i f o r m s u p e r c o i l of 45-50 A a v e r a g e p i t c h a n d 100 A o u t e r d i a m e t e r . T h e P a r d o n et al. m o d e l is b a s e d e n t i r e l y on t h e o c c a s i o nal o b s e r v a t i o n of a l l 0 A X - r a y r e f l e c t i o n in gels a n d fibers of n u c l e o h i s t o n e . T h i s s p a c i n g is n o t o b s e r v e d at n u c l e o h i s t o n e c o n c e n t r a t i o n s less t h a n 15 p. c e n t ; h o w e v e r , w h e n t h e n u c l e o h i s t o n e is p r e c i p i t a t e d b y t h e a d d i t i o n of 10 .2 M d i v a l e n t salt t h e 110 A s p a c i n g a p p e a r s E7]. A c o n s i d e r a t i o n of t h o s e c o n d i t i o n s u n d e r w h i c h this s p a c i n g d o e s a p p e a r h a s led b o t h L u z z a t i a n d N i c o l a i e f f ~8] a n d B r a m [5] to c o n c l u d e t h a t it r e s u l t s f r o m t h e s i d e b y side a g g r e g a t i o n of i n d i v i d u a l p a r t i c l e s 100 A in d i a m e t e r . I n a n y case, t h e r e s u l t s p r e sented here should show that the very open struct u r e of this m o d e l is not c o n s i s t e n t w i t h X - r a y s c a t t e r i n g in s o l u t i o n n o r w i t h r e s o l u t i o n e l e c t r o n microscopy. T h e f e a t u r e s of t h e X - r a y fiber d i f f r a c t i o n patt e r n [I] c a n also be w e l l e x p l a i n e d b y t h e aggre68
Stanley Bram.
1006
gation or h i g h e r o r d e r f o l d i n g of a 45 A p i t c h s u p e r coil. The 110 A and p e r h a p s also the 55 A s p a c i n g s could be due to the axial p r o j e c t i o n s w h i c h c o m p r i s e about 20 p. cent of the nucleohistone m a t e r i a l o b s e r v e d in e l e c t r o n m i c r o g r a p h s [3, 6!. These p r o j e c t i o n s at times a p p e a r to be a
t h i n sections d i s p e r s e d n u c l e o h i s t o n e w a s c e n t r i fuged at 150.000 g for 18 h o u r s and the pellet was fixed in f o r m a l i n a n d t h e n for seven m i n u t e s in 1 p. cent Oso 4 in 0.1 M p h o s p h a t e buffer at pH 7. T h e pellet w a s w a s h e d t h r o u g h l y w i t h distilled w a t e r and e m b e d d e d in E p o n - A r a l d i t e [10]. T h i n sections w e r e s t a i n e d i n 7 p. cent u r a n y l - m a g n e slum acetate for two h o u r s and then lead citrate for 15 m i n u t e s ~ll~. The section w e r e m a d e and e x a m i n e d in the l a b o r a t o r y of Dr H. Ris of the U n i v e r s i t y of W i s c o n s i n . F o r negative staining, a drop of solution was p l a c e d on a c a r b o n coated grid (excess solution was r e m o v e d ~ ' i t h filter p a p e r s and then floated on 2 p. cent u r a n y l f o r m a t e for several minutes). T h e grid w a s then d r i e d by t o u c h i n g one edge to filter paper. The X-ray s c a t t e r i n g m e t h o d s h a v e been prev i o u s l y d e s c r i b e d [5, 6].
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Fro. 1. - - Sketches of ,two nudeohistone models. The Bram and Ris model is a non-uniform coil having an average pitch of 5'0 A but for convenience is drawn here as a regular coil.
loop of I h e 100 A fiber folded u p o n itself. The s t r o n g equatorial reflections at 60 and 30 A w o u l d be e x p e c t e d f r o m the i n t e r p a r t i c l e spacings of m o l e c u l e s h a v i n g a cross-section r a d i u s of gyration of 30 A. T h e 38 and 22 A reflections w o u l d then resnlt f r o m the average p a r a m e t e r s of the ind i v i d u a l particles. Spacings at 40 and 23 A are o b s e r v e d in the X-ray s c a t t e r i n g p a t t e r n of i n d i v i d u a l molecules in solution and gels ; these spacings and t h e i r relative intensities c o r r e s p o n d w e l l w i t h those e x p e c t e d f r o m the m o d e l of Brain and Ris [6].
MATERIAL AND METHODS. N u c l e o h i s t o n e was isolated f r o m calf t h y m u s e m p l o y i n g a m o d i f i c a t i o n of the Z u b a y a n d Doty p r o c e d u r e [9]. The m e t h o d i n v o l v e s the use of m u c h less s h e a r and an isolation pH of 6.5 i n s t e a d of 8.0 [5, 6]. N u c l e o h i s t o n e isolated at p H 8.0 h a d a p o o r l y defined outline in e l e e t r o n m i c r o g r a p h s and e x h i b i t e d m u c h m o r e side by side aggregation than m a t e r i a l isolated at pH 6.5 [51. F o r study in
BIOCHIMIE, 19,72, 54, n ° 8.
T H E NUMBER OF DNAs P E R CROSSECTION T h e d i a m e t e r of n u c l e o h i s t o n e fibers is occasionally o b s e r v e d to be as small as 25 A in stretches contiguous w i t h 80-10,0 A t h i c k r e g i o n s (see fig. 2b). This observation, plus p a r t i a l p r o n a s e digestion studies of n u c l e o h i s t o n e on e l e c t r o n mic r o s c o p e grids [3], r e q u i r e that there be only one DNA double h e l i x p e r cross section, coiled or folded to m a k e a fiber 100 X in diameter. I n c o n t r o vertible e v i d e n c e for a s u p e r coiled s t r u c t u r e is the r e v e r s i b l e a p p e a r a n c e of a well defined DNA X-ray p a t t e r n and the d i s a p p e a r a n c e of the 38-40 X spac i n g u p o n the s t r e t c h i n g of well o r i e n t e d nuc]eoh i s t o n e fibers [4]. SMAEL AND INTE,RM~,DIATE ANGLE X-RAY SCATTERING. The entire n u c l e o h i s t o n e X-ray s c a t t e r i n g pattern w a s f o u n d to be i n v a r i a n t o v e r at least the r a n g e of i o n i c strength b e t w e e n 0.8 and 20 mM s o d i m n p h o s p h a t e at pH 6.8 [5, 6]. F r o m the small angle X-ray s c a t t e r i n g w e h a v e o b t a i n e d a cross section r a d i u s of g y r a t i o n of 30 A and a mass p e r unit length of 12010 Daltons/3~ [6] w h i c h are in r e a s o n a b l y good a g r e e m e n t w i t h the values of 26 A and 150,0 Daltons/3~ o b t a i n e d by Luzzati and Nicolaieff [8]. T h e cross section r a d i u s of gyration Re, is defined as the root m e a n s q u a r e dist a n c e of electrons f r o m the long axis. F o r a structure c o n s t r u c t e d out of one 25 A thread, a R e of 30 A r e q u i r e s that its c e n t e r be at an a v e r a g e of 28 A f r o m the axis. Such a s t r u c t u r e is r a t h e r c o m p a c t as the c e n t e r <
A structural chromosome w i d e (see fig. 1 a). T h e 30 A e x p e r i m e n t a l c r o s s s e c t i o n r a d i u s of g y r a t i o n a g r e e s w e l l w i t h t h e diameter observed in the electron microscope but n o t at all w i t h t h e 50 A c r o s s s e c t i o n r a d i u s o f g y r a t i o n e x p e c t e d f r o m t h e P a r d o n el al. m o d e l .
model.
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experimental M/L represents a minimum vahm, f o r if e x t e n s i v e s i d e b y s i d e a g g r e g a t i o n w e r e p r e sent the e x p e r i m e n t a l result w o u l d be less t h a n t h e t r u e v a l u e o f t h e i s o l a t e d p a r t i c l e s . But f r o m t h e m i n i m u m v a l u e of t h e m a s s p e r u n i t l e n g t h
FifunE 2; a, b, and e are thin sections of an embedded nucleohistone pellet positively stained w i t h lead citrate and uranyl acetate. FI6. 2 a. - - <> denotes a region w h e r e the folding of a 25 A d i a m e t e r fiber is particularly visible, and <> marks a region showing b a n d s of contrast 25 A thick about 40.-50 A apart. × 300,000. 2 b. - - A thin fiber, 20.-25 A in diameter, continuous w i t h a thicker fiber noted by < × 267,000. 2 c. - - The compact fibrous u l t r a s t r u e t u r e of nucAeohistone is seen. × 333,900. 2 d. - - A11igned nucleohistonc fibers negatively stained w i t h uranyl formate. P o o r penetration of stain into the fiber blurs =the image b u t the fine structure appears similar to t h a t with positive staining × 1,60',000.
The mass per unit length contains information about the d i s t r i b u t i o n of mass in the d i r e c t i o n of l h e l o n g axis. T h e m a s s p e r u n i t l e n g t h c o n t r i b u t i o n of t h e DNA i n n u c l e o h i s t o n e is 3-4 t i m e s t h a t of i s o l a t e d B-DNA (18,6 D a l t o n s / A ) ; t h e f a c t t h a t t h e r e is o n l y o n e DNA p e r c r o s s s e c t i o n r e q u i r e s t h a t t h e DNA b e f o l d e d o r s u p e r c o i l e d to give a 3-4 f o l d c o n t r a c t i o n f r o m its e x t e n d e d l e n g t h . T h e
BIOCHIMIE, 1972, 54, n ° 8.
one can calculate a m a x i m u m value of the s u p e r coil p i t c h . Ii c a n b e s h o w n t h a t t h e p i i c h m u s t be s m a l l e r t h a n a b o u t 60 A, a n d a v a l u e o f 50 A w o u l d be in better a g r e e m e n t w i t h the results
~5, 6j. Independent information concerning the shape of l h e n u c l e o h i s t o n e p a r t i c l e is o b t a i n a b l e f r o m t h e X - r a y s c a t t e r i n g p a t t e r n at i n t e r m e d i a t e s c a t -
Stanley Brain.
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t e r i n g angles. In this region of the scattering p a t t e r n the shape of the curve is a f u n c t i o n of the shape of the particle. Two m a x i m a in the plot of the i n t e n s i t y scattered vs, scattering angle are observed, one at an equivalent bragg s p a c i n g of 4.0 A and the other at 22 A. W h e n the scattering p a t t e r n is c o m p a r e d to curves calculated for various type of models, coils were f o u n d to give the best agreement [5~. W i t h a super coil model h a v i n g an R e of 3,0 A and a p i t c h of 45 A an exact fit in both the s p a c i n g a n d relative intensities of the e x p e r i m e n t a l curve was obtained. However, the e x p e r i m e n t a l m a x i m a were m u c h b r o a d e r t h a n those calculated for a regular helix. This implies that the coiling is not regular. W h e n nlodel structures featuring a v a r i a t i o n i n r a d i u s a n d pitch were employed a good fit to the X-ray scattering was o b t a i n e d [5]. ELECTRON MICROSCOPY. My m a i n objective i n p r e s e n t i n g these micrographs is to show that the t e r t i a r y s t r u c t u r e of n u c l e o h i s t o n e is compact, a n d i m p e n e t r a b l e to p r o t e i n molecules. For example, a small p r o t e i n such as lysozyme has a diameter of 50 A and the lac repressor w h i c h is active as a t e t r a m e r [12] w o u l d have a m u c h larger diameter. I suggest that the r e a d e r i m a g i n e a 6,0 A sphere (which is about 2/3 the diameter of the fibers) and ask w h e t h e r or not this sphere w o u l d p e n e t r a t e into the i n t e r i o r of the images. By this c r i t e r i a the model of Brain a n d Ris is a very compact s t r u c t u r e - - a sphere 30 A in diameter w o u l d not p e n e t r a t e the gyres of the super coil. I n fact, the spaces b e t w e e n the gyres of this model are sufficiently small to allow for the histones of n e i g h b o r i n g gyres to form h y d r o p h o b i c linkages and to t h e r e b y stabilize the conformation. Figure 2 shows high resolution electron micrographs of calf t h y m u s n u c l e o h i s t o n e e m b e d d e d in t h i n epoxy sections. The n u c l e o h i s t o n e has the same general a p p e a r a n c e as in sections of whole nuclei [13]. This n u c l e o h i s t o n e [6] also r e t a i n s the m o r p h o l o g y of c h r o m a t i n fibrils o b t a i n e d by s p r e a d i n g open whole cells on an air salt solution i n t e r p h a s e [3, 6~. The 10,0 A fibers are seen to be c o n s t r u c t e d out of a t h i n n e r fiber 20-30 A i n diameter w h i c h folds up to make the t h i c k e r fiber. At times the folding can be r e a d i l y followed (see figure 2 a a n d c). Bands of contrast r u n n i n g at large angles to the axis are often observed ; they sometimes have the a p p e a r a n c e of gyres i n a coil. The distance b e t w e e n these b a n d s is 40-50 A, w h i c h is in agreement w i t h the X-ray results. However, the structure is usually not r e g u l a r - -
BIOCHIMIE, 19,72, 54, n ° 8.
that is, some regions are more folded t h a n others. But the most i m p o r t a n t observation is that the u l t r a s t r u c t u r e appears to be quite compact. Very few regions are seen w h e r e a 60 A sphere could penetrate, a n d i n general the i n t e r i o r of the substructure appears to be inaccessible to molecules larger t h a n 30 to 40 A. Such a c o m p a c t folding was also observed b y Wettstein a n d Sotelo i n their sections of n u c l e i [13]. I n figure 2 b a rare stretch of u n f o l d e d chromatin is noticed. These u n f o l d e d regions make up at most a few p e r c e n t of the total mass, w h i c h disagrees w i t h the extensive u n f o l d i n g p r o p o s e d by Clark and Felsenfeld [14]. Nucleohistone was also s t u d i e d by negative staining. U n f o r t u n a t e l y , m u c h of the u l t r a s t r u c t u r e is b l u r r e d by the i m p a r t i a l p e n e t r a t i o n of the particles of the u r a n y l formate p r e c i p i t a t e w h i c h are about 15 to 25 A i n diameter. Such a compact structure w h i c h i m p e d e s the p e n e t r a t i o n of these small particles should limit the access of p r o t e i n molecules to the DNA. These e l e c t r o n m i c r o g r a p h s are not at all consistent with the loose super helix of P a r d o n et al. [4] but s u p p o r t the compact structure of the Brain and Bis model.
Wide angle X-rag scattering. The difference between the s e c o n d a r y structure of DNA in solution and in n u c l e o h i s t o n e could be the reason for the t e r t i a r y super coiling of the DNA. Wide angle X-ray scattering e x p e r i m e n t s [15] show that the t u r n angle per base p a i r of the DNA in n u c l e o h i s t o n e is about 10 p. cent larger t h a n i n solution. That is if one assumes that DNA in solution has 10 base p a i r s per t u r n , there are about 9 base pairs p e r repeat i n nucleohistone. If the ends of a DNA region were held fixed, a change in the s e c o n d a r y w i n d i n g w o u l d cause t e r t i a r y s u p e r coiling i n complete analogy to closed c i r c u l a r DNA [15]. F i x a t i o n could be achieved b y p r o t e i n cross links or m u l t i p l e b i n d i n g to the n u c l e a r m e m b r a n e . The i n c r e a s e d right h a n d e d w i n d i n g of a DNA region i n n u c l e o h i s t o n e w o u l d result i n a left h a n d e d super coiling p r o p o r t i o n a l to the change i n w i n d i n g . It has been s h o w n that the 10 p. cent difference b e t w e e n the t u r n angle of DNA i n solution a n d of ~be b u l k DNA in nuclcohistone is consistent w i t h the compact s u p e r coil of tile Bram and Ris model [15]. Thus, the cross seclion r a d i u s of g y r a t i o n a n d mass per u n i t length, i n t e r m e d i a t e angle scattering maxima, the wide angle-X-Ray p a t t e r n a n d elect r o n m i c r o s c o p y all i n d e p e n d e n t l y agree w i t h a compactly folded strncture h a v i n g an average p i t c h of 50 A.
A structural chromosome
RECOGNITION DNA. The results also show that the super coiling is n o n - u n i f o r m . This is a c o n c l u s i o n w h i c h must also be d r a w n from the fact that the various histone fractions differ i n their ability to super coil DNA. F o r example, r e m o v i n g very lysine rich histonc I from n u c l e o h i s t o n e or a d d i n g it to DNA causes no change in the t e r t i a r y structure [16], but the a d d i t i o n of a r g i n i n e r i c h histone results i n the super coiling of the DNA [17]. I have previously s h o w n by w i d e angle scattering experiments that DNA very r i c h in AT base pairs has a s e c o n d a r y structure different from DNA of lower AT content, and have proposed that very AT r i c h DNA serves as m a r k e r s for control a n d recognition [18]. Since h i s t o n e I b i n d s p r e f e r e n t i a l l y to DNA w h i c h is r i c h e r in AT [19], this type of DNA might be kept relatively u n f o l d e d due to the weak s u p e r coiling p o w e r of this histone. Its u n i q u e s e c o n d a r y structure m i g h t also p r e v e n t if from f o r m i n g a tight super coil. I p r o p e s e that - - except for a small a m o u n t of DNA serving a r e c o g n i t i o n role - - the DNA i n chromosomes is tightly folded a n d therefore inaccessible to r e c o g n i t i o n or translation proteins. This m e c h a n i s m w o u l d facilitate the f i n d i n g of a correct r e c o g n i t i o n site in the chromosomal mass, as only a small p o r t i o n of the DNA could be sampled. Crick has p r o p o s e d that the DNA used for recognition is tightly folded, and that w h i c h codes for p r o t e i n is extended [2~)]. The model proposed here is i n some ways the o p p o s i t e : r e c o g n i t i o n DNA represents a m i n o r fraction a n d is relatively extended w h i l e coding DNA r e m a i n s tightly folded (and at the same time o v e r t u r n e d ) unless it is activated by a l o o s e n i n g of the t e r t i a r y and secondary structure. F o r the model as proposed, it is not necessary to c o m m e n t on the relative a m o u n t s of c o d i n g to control ])NA. P h y s i c a l chemical studies of inactive chromatin i m p l y that most, if not all, of the c o n s t i t u e n t DNA is double s t r a n d e d [4, 6, 9, 15] a n d i n i t i a l r e c o g n i t i o n would be expected to be of double s t r a n d e d DNA as is the case for the lac a n d phage repressor [21, 22]. Specificity could be achieved by two or three short b i n d i n g regions of different s e c o n d a r y structure separated by a requisite n u m b e r of base pairs. But it is more reasonable to suggest that r e c o g n i t i o n involves a two step process : first the semi-specific b i n d i n g to a general type of DNA, followed b y a r e c o g n i z e r induced modification of the s e c o n d a r y structure. Only the correct D~NA site will be distorted so as to give a u n i q u e strong b i n d i n g configuration with BIOCHIMIE, 1972, 54, n ° 8.
1009
model.
a p r o d u c t i v e fit. I have p r e v i o u s l y suggested that these general r e c o g n i t i o n regions are very r i c h i n AT [151 ; c o n s e q u e n t l y very AT r i c h DNA could competed for recognizers by n o n - p r o d u c t i v e binding as has been s h o w n for the lac repressor [see reference 30]. To be t r a n s c r i b e d coding DNA must u n w i n d , at least partialy, w h i c h w o u l d r e q u i r e an equivalent w i n d i n g of the n e i g h b o r i n g DNA. The adjacent DNA is c o n s t r a i n e d from f u r t h e r w i n d i n g by its histone i n d u c e d compact s e c o n d a r y structure ; however, d e n a t u r a t i o n or over w i n d i n g of "the adjacent r e c o g n i t i o n site w o u l d remove this const r a i n t a n d the coding DNA could u n w i n d for transcription.
P R O P E R T I E S OF VERY AT RICH DNA. lit is p e r t i n e n t to c o n s i d e r w h a t we k n o w about the s e c o n d a r y structure of very AT r i c h DNA. Figure 3 shows the wide angle s c a t t e r i n g p a t t e r n s of a series of I)NAs of different base c o m p o s i t i o n in .05 NaCl. Up to about 60 p. cent AT the wide
~
~ :
6,0 0,10
0,15
0,20
$¢atterin 9 angle 15,4
10,3
0,25
' ) 0,30
6,2
5,2
radiorts
7,7 o Sp|A)
FIG. 3. The wide angle scattering patterns for solutions of DNA from E. colt (E):, M. lysodeikiticus (M), calf .thymus (T), Cl. Perfringens (C) and B. cereus (B) in. 05M NaC1. (After reference [181). -
-
angle p a t t e r n s are similar, but at about 65 p. cent AT (A + T / G + C : 2.0) a significant change occurs ; all of the m a x i m a are c o n s i d e r a b l y flattened, especially the one near 13 A, a n d the maxim u m at 10 A is significantly i n c r e a s e d in i n t e n s i t y c o m p a r e d to the first. Thus the s e c o n d a r y structure must be different from that of DNA of typical base c o n t e n t i n solution. Recent X-ray fiber
Stanley Brain.
1010
d i f f r a c t i o n e x p e r i m e n t s s h o w that v e r y AT r i c h DNA in fibers adopts several n e w f o r m s w h i c h are not s h o w n by t y p i c a l DNA [23, 24]. T h i s polym o r p h i s m is consistant w i t h the r e c o g n i t i o n function p r o p o s e d for AT r i c h DNA. On the basis of some r a t h e r scanty X-ray data for AT r i c h DNA a n d f r o m s t e r i o c h e m i c a l cons i d e r a t i o n s I suggested that v e r y AT r i c h DNA m i g h t not a d o p t the A f o r m [18], and s u b s e q u e n t X-ray fiber d i f f r a c t i o n studies in o u r l a b o r a t o r y s u p p o r t this suggestion [24], DNA that is r i c h e r t h a n about 65 p. cent AT has not been f o u n d to adopt the A form, u n d e r c o n d i t i o n s w h i c h give this c o n f o r m a t i o n for DNA less r i c h in AT. This
is so for those that have been c h a r a c t e r i s e d in procaryotes : the lac o p e r a t o r [30] - - and)~ prom o t o r [31] (*). It w o u l d be in a g r e e m e n t w i t h the m o d e l if c y t o p l a s m i c RNA f r o m h i g h e r cells w e r e of l o w e r AT c o n t e n t t h a n the bulk DNA. I n d e e d , this seems the case for those R NAs w h o s e base contents h a v e been d e t e r m i n e d (see table I). T h e h i g h GC c o n t e n t of h i s t o n e m e s s e n g e r is p a r t i c u l a r l y n o t e w o r t h y , for h i s t o n e c o n t a i n s large amounts of l y i s n e w h i c h is c o d e d f o r by e i t h e r AAA or AAG, both of w h i c h substantially i n c r e a s e the AU c o n t e n t of the m e s s e n g e r ; the messenger, h o w e v e r is GC rich. T h e h i g h (~C c o n t e n t of these c y t o p l a s m i c RNAs could also be due to the prefer e n t i a l f o r m a t i o n of double s t r a n d e d r e g i o n s
TABLE I.
Base compositions of cytoplasmic RNAs of higher organisms (1). A+U
RNA
Calf eye lens messenger (32) . . . . . . . . . . . . . . . . . . . . . . Hemoglobin messenger (33) . . . . . . . . . . . . . . . . . . . . . . . Calf histone messenger (34) . . . . . . . . . . . . . . . . . . . . . . . Xenopus (35) 5 S RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transfer HNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 S ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 S ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bulk DNA
A+T G+C
0,97 0,71 0,88 1.3tol.4
0,75 0,67 0,85 0,59
(1) The possible presence, of poly A in the messengers has not. been accounted for, however it could only rncrease the GC content of the remainder. is also i n f e r r e d f r o m i n f r a r e d studies [251. (it is w o r t h m e n t i o n i n g h e r e that although p o l y [d(A-T)] • p o l y [d(A-T)] does a d o p t the A f o r m [263, p o l y (dA) • p o l y (dT) does not [27]).The i d e a that the B-A t r a n s i t i o n is i n v o l v e d in t r a n s c r i p t i o n w a s p r o p o s e d by c r y s t a l l o g r a p h e r s because the RNA-DNA h y b r i d is f o u n d only in the A-form [28]. T h a t t r a n s c r i p t i o n r e q u i r e s a B-A t r a n s i t i o n is strongly i m p l i e d f r o m r e c e n t studies s h o w i n g that the c i r c u l a r d i c h r o i s m s p e c t r u m of the RNA polym e r a s e - s i g m a b i n d i n g site goes f r o m the B to the A t y p e s p e c t r u m u p o n starting RNA synthesis [29]. If a B to A t r a n s i t i o n must p r e c e d e t r a n s c r i p t i o n , v e r y AT r i c h DNA in h i g h e r o r g a n i s m s m i g h t not be t r a n s c r i b e d w i t h the usual t r a n s l a t i o n system d e s i g n e d for t y p i c a l DNA b e c a u s e it does not adopt e i t h e r the B or A form.
which would attack.
be
resistent
to
certain
nuclease
It w o u l d be w o r t h w i l e in studies of s p a c e r and satellile DNAs to i n v e s t i g a t e w h e t h e r or not the g e n e t i c code has a r e s t r i c t e d r a n g e of a l l o w a b l e base contents and sequences. In a d d i t i o n to the a d v a n t a g e of setting aside classes of DNA to serve roles o t h e r than coding, these c r i t e r i a w o u l d provide a m e a n s of l i m i t i n g the a p p e a r a n c e in m-RNA of the n o n c o d i n g t e r m i n a t i o n codons UAG and UAA w h i c h are m o r e than 66 p. cent AU.
Acknolwledgements. I thank David Perrin for his interest and many helpful suggestions. These ideas were presented at the Colloquium of the Socidt6 Chimie Biologique in Strasbourg, March 1972.
SOME P R E D I C T I O N S OF T H E MODI~L. The m o d e l p r e d i c t s that o p e r a t o r and p r o m o t e r sites s h o u l d be in v e r y AT r i c h regions, a n d this
BIOCHIMIE, 1972, 54, n ° 8.
(*) I't has recently been found tha,t the RNA polymerase binding sites of phage fd replicative form DNA are very AT rich and are also double stranded [36].
A s t r u c t u r a l c h r o m o s o m e model. RfiscM~. Le mod61e p r o p o s 6 p e u t ~tre r 6 s u m 6 c o m m e s u i t : 1) La p l u s g r a n d e p a r t i e de t ' A D N d a n s les c h r o m o s o m e s e s t n o n u n i f o r m 6 m e n t r e p l i 6 e en u n e n r o u i c m e n t t e r t i a i r e c o m p a c t g a u c h e d o n t le p a s m o y e n et le r a y o n s o n t de 45 .~. P o u r ce q u i e s t de la s t r u c t u r e s e c o n d a i r e le pas, o u e n c o r e ]e r a y o n de cet ADN e s t de 10 p. c e n t p l u s p e t i t q u ' e n s o l u t i o n . 2) E n v e r t u de cette s t r u c t u r e s e c o n d a i r e r e n v e r s 6 e , la m a j e n r e f r a c t i o n de cot ADN n e p e u t 6tre t r a d u i t . C o n c u r r e m m e n t , la s t r u c t u r e t e r t i a i r e c o m p a c t e b l o q u e ~l'acc6s d e s m o l 6 c u l e s de r e c o n n a i s s a n c e et d e p o l y m 6 r a s e h c~t ADN. 3) Seule u n e p e t i t e f r a c t i o n de I'ADN t o t a l n ' e s t p a s 6 t r o i t e m e n t repli4e ct e s t p a r c o n s 6 q u e n t u t i l i s a b l e p o u r la r e c o n n a i s s a n c e . I1 est s u g g 6 r 6 q u e cet ADN de r e c o n n a i s s a n c e a u n e c o m p o s i t i o n en b a s e s n o n t y p i q u e et q u ' e l l e est tr6s r i c h e en AT. L ' A D N de r e c o n n a i s s a n c e est c o n s e r v 6 n o n r e p l i 4 e p a r sa s t r u c t u r e s e c o n d a i r e u n i q u e on s o n i n t e r a c t i o n p r 6 f 6 r e n t i e l l e avec les h i s t o n e s I. La r e c o n n a i s s a n c e spScifique p e u t i m p l i q u e r la l i a i s o n h u n t y p e g 6 n 6 r a l d ' A D N (tr6s r i c h e en AT) s u i v i e p a r n n e t r a n s i t i o n de s t r u c t u r e i n d u i t e p a r la mo16cnle p o u r p e r m e t t r e la r e c o n n a i s s a n c e e x a c t e de la s 6 q n e n c e p r i m a i r e . 5) I1 est prop.os6 q u e d a n s ]a cellule, I'ADN tr6s r i c h e e n AT ne p e u t c o m m e n c e r la t r a n s c r i p t i o n de p a r sa s t r u c t u r e s c c o n d a i r e r e m a r q u a b l e et p a r c e q u ' i l n ' a d o p t e p a s la f o r m e A.
ZUSAMMENFASSUNG. D a s v o r g e s c h l a g e n e Modell k a n n w i e f o l g t z u s a m m c n g e f a s s t w e r d e n : 1. D e r g r 6 s s t e Tell d e r DNS in d e n C h r o m o s o m c n w i r d u n g l e i c h m i i s s i g in e i n e r k o m p a k t e n tertHiren linken Struktur, dcren mittlerer Windungsa b s t a n d u n d R a d i u s 45 .~ be,t~iigt, z u s a m m e n g e f a l t e t . Bet d e r s c k u n d i i r e n S ~ r u k t u r is.t die W i n d u n g s H i n g e o d e r a u c h d e r R a d i u s u m 10' p. c e n t k l e i n e r a l s in LSsung. 2)Wegen dieser umgekehrten sekundiiren Struktur k a n n d i e g r S s s t e F r a k t i o n d i e s e r DNS n i c h t u m g e s e t z t w e r d e n . Z u s a m m e n w i r k e n d v c r h i n d e r t die k o m p a k t e tertiiire Struktur den Zutritt der Rekognoszierungsm o l e k i i l e u n d d e r P o l y m e r a s e z u d i c s e r DNS. 3. A l l e i n e i n c g e r i n g e F r a k t i o n d e r g e s a m t e n DNS w i r d n i c h t e n g g e f a l t e t u n d ist f o l g l i c h fiir d i e R e k o gnoszierung verwendbar. Es wird angenommen, dass diese Rekognoszierung-DNS sich aus nicht-lypischen B a s e n z u s a m m e n s e t z t u n d d a s s sic r e i c h a n AT ist. Die R c k o g n o s z i e r n n g s - D ' N S f a l t e t sich n i c h t d u r c h i h r e b l o s s e sekund~ire S t r u k t u r o d e r d u r c h i h r c v o r z u g s w c i s e W e e h s e B v i r k u n g m i t d e n H i s t o n e n . I. Die s p e z i fische R e k o g n o s z i e r u n g k a n n die B i n d u n g a n e i n e m a l l g e m e i n e n , a n AT s e h r r e i c h e n T y p d c r DNS, die y o n e t h e r S t r u k t u r i i n d c r u n g , die d u r c h d a s Molekiil v o r g e n o m m e n w i r d , u m die g e n a u e R e k o g n o s z i e r u n g der primiiren Sequenz zu erlauben, implizieren. 4. Es w i r d a n g e n o m m e n , d a s s i n de.r ZeHe d i e a n AT s e h r r.eich.e DNS die T r a n s k r i p t i o n a u f G r u n d i h r e r
BIOCHIMIE, 1972, 54, n ° 8.
1011
b e a c h t l i c h e n s e k u n d ~ i r e n S t r u k t u r , da sie die F o r m A nicht annimmt, nicht beginnen kann. REFERENCES. 1. R i s H. (1957)
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Chemical
Basis
The ihnction of the structure of DNA in chromosomes. S t a n l e y BRAI~I.
D~partement de Biologie Mol~culaire, lnstitut Pasteur, Paris 15, France. (3/6/1972).
Summary. - - The proposed model can be summarized as follows : 1) Most of the DNA in chromosomes is non-uniformly folded into a compact left handed super coil whose average pitch and radius are 45 A. The pitch or radius of this double helical DNA are 10 p. cent smaller than in solution. 2) By virtue of its compact secondary structure this m a j o r fraction of the D NA cannot be transcribed ; concurrently, the compact tertiary structure blocks the access of recognition ~and polymerase molecules to this DNA. 3) A smal:l fraction of the total DNA is not tightly folded and is thereby marked for recognition. It is suggested that this recognition DNA is of non-typical base composition and that it is very rich in AT. The recognition DNA is kept unfolded by its unique secondary structure and by preferential interaction with lysine rich histories. 4) Specific recognition may involve the less specific binding to a general type of DNA (very AT rich DNA) followed by a recognizer-induced transition to allow for productive binding to the proper sequence. 5) It is proposed thai in the cell very AT rich DNA cannot initiate transcription due to its unique secondary structure and because it does not adopt the A-form.
INTRODUCTION. A m o d e l is p r o p o s e d h e r e for t h e s t r u c t u r e a n d f u n c t i o n of D N A in c h r o m o s o m e s of h i g h e r o r g a n i s m s . T h e m o d e l is b a s e d u p o n s t r u c t u r a l s t u d i e s of n u c l e o h i s t o n e a n d D NA. It w i l l p r o v i d e n e i t h e r a c o m p l e t e n o r e x a c t d e s c r i p t i o n of t h e c h r o m o some. My p u r p o s e is to a t t e m p t to g i v e a f r a m e of r e f e r e n c e w h i c h w i l l a i d in t h e r e l a t i n g of s t r u c t u r a l s t u d i e s of c h r o m o s o i n e s a n d n u c l e i c a c i d s "to t h e f u n c t i o n of DNA. T h e f u n c t i o n of t h e s t r u c t u r e of t h e c h r o m o s o m e is to e f f i c i e n t l y h a n d l e l o n g D N A m o l e c u l e s . It p r o v i d e s for o r d e r e d t r a n s c r i p t i o n , r e p l i c a t i o n a n d m i t o t i c s e g r e g a t i o n . T h e c h r o m o s o m e c o n s i s t s of D,NA i n t i m a t e l y a s s o c i a t e d w i t h a b a s i c p r o t e i n - h i s t o n e - - a n d v a r i a b l e a m o u n t s of n o n h i s t o n e a c i d p r o t e i n a n d RNA. T h e D N A - h i s t o n e c o m p l e x w h i c h w e call n u c l e o h i s t o n e is r e p r e s s e d w i t h resp e c t to p r o t e i n s y n t h e s i s . T h a t is, it m a k e s v e r y little or no m - R N A w i t h R N A p o l y m e r a s e in vitro, a n d it is t h e d o m i n a n t f o r m of c h r o m a t i n in m e t a b o l i c a l l y i n a c t i v e cells. N u c l e o h i s t o n e is o r g a n i z e d i n t o fibers t h a t fold o r coil up to m a k e t h e c o n d e n sed c h r o m o s o m e . C h r o m o s o m e s in t h e c o n d e n s e d state h a v e a d i a m e t e r on t h e o r d e r of 2 00010 000 A a n d e l e c t r o n m i c r o s c o p i s t s [1, 2] h a v e s h o w n t h a t t h e y c o n s i s t of fibers 200-250 A in diam e t e r . Ris has s h o w n t h a t if t h e 20'0 A fibrils a r e t r e a t e d w i t h c h e l a t i n g agents to r e m o v e c a l c i u m t h e y a r e g r a d u a l l y t r a n s f o r m e d i n t o fibers 80-100 A in d i a m e t e r [3]. He suggests t h a t t h e 200 A fiber
c o n s i s t s of 100 A fibers f o l d e d b a c k u p o n t h e m s e l v e s or a g g r e g a t e d to m a k e t h e t h i c k e r fiber. It is t h e 100 A n u c l e o h i s t o n e fiber w h i c h m o s t w o r k e r s c o n s i d e r to be t h e b a s i c c h r o m o s o m a l u n i t a n d h e r e I w i s h to r e l a t e its t e r t i a r y , s e c o n d a r y , a n d p r i m a r y s t r u c t u r e to t h e c o n t r o l of gene a c t i v i t y . T w o d e t a i l e d s t r u c t u r a l m o d e l s for n u c l e o h i s t o n e h a v e b e e n p r o p o s e d - t h a t of P a r d o n , W i l k i n s a n d R i c h a r d s [4] a n d t h a t of B r a m a n d Ris. [5, 6] (see f i g u r e 1). T h e f o r m e r is a l o o s e s u p e r c o i l of p i t c h 120 A a n d o u t e r d i a m e t e r 130 A. T h e o t h e r is a c o m p a c t n o n - u n i f o r m s u p e r c o i l of 45-50 A a v e r a g e p i t c h a n d 100 A o u t e r d i a m e t e r . T h e P a r d o n et al. m o d e l is b a s e d e n t i r e l y on t h e o c c a s i o nal o b s e r v a t i o n of a l l 0 A X - r a y r e f l e c t i o n in gels a n d fibers of n u c l e o h i s t o n e . T h i s s p a c i n g is n o t o b s e r v e d at n u c l e o h i s t o n e c o n c e n t r a t i o n s less t h a n 15 p. c e n t ; h o w e v e r , w h e n t h e n u c l e o h i s t o n e is p r e c i p i t a t e d b y t h e a d d i t i o n of 10 .2 M d i v a l e n t salt t h e 110 A s p a c i n g a p p e a r s E7]. A c o n s i d e r a t i o n of t h o s e c o n d i t i o n s u n d e r w h i c h this s p a c i n g d o e s a p p e a r h a s led b o t h L u z z a t i a n d N i c o l a i e f f ~8] a n d B r a m [5] to c o n c l u d e t h a t it r e s u l t s f r o m t h e s i d e b y side a g g r e g a t i o n of i n d i v i d u a l p a r t i c l e s 100 A in d i a m e t e r . I n a n y case, t h e r e s u l t s p r e sented here should show that the very open struct u r e of this m o d e l is not c o n s i s t e n t w i t h X - r a y s c a t t e r i n g in s o l u t i o n n o r w i t h r e s o l u t i o n e l e c t r o n microscopy. T h e f e a t u r e s of t h e X - r a y fiber d i f f r a c t i o n patt e r n [I] c a n also be w e l l e x p l a i n e d b y t h e aggre68
Stanley Bram.
1006
gation or h i g h e r o r d e r f o l d i n g of a 45 A p i t c h s u p e r coil. The 110 A and p e r h a p s also the 55 A s p a c i n g s could be due to the axial p r o j e c t i o n s w h i c h c o m p r i s e about 20 p. cent of the nucleohistone m a t e r i a l o b s e r v e d in e l e c t r o n m i c r o g r a p h s [3, 6!. These p r o j e c t i o n s at times a p p e a r to be a
t h i n sections d i s p e r s e d n u c l e o h i s t o n e w a s c e n t r i fuged at 150.000 g for 18 h o u r s and the pellet was fixed in f o r m a l i n a n d t h e n for seven m i n u t e s in 1 p. cent Oso 4 in 0.1 M p h o s p h a t e buffer at pH 7. T h e pellet w a s w a s h e d t h r o u g h l y w i t h distilled w a t e r and e m b e d d e d in E p o n - A r a l d i t e [10]. T h i n sections w e r e s t a i n e d i n 7 p. cent u r a n y l - m a g n e slum acetate for two h o u r s and then lead citrate for 15 m i n u t e s ~ll~. The section w e r e m a d e and e x a m i n e d in the l a b o r a t o r y of Dr H. Ris of the U n i v e r s i t y of W i s c o n s i n . F o r negative staining, a drop of solution was p l a c e d on a c a r b o n coated grid (excess solution was r e m o v e d ~ ' i t h filter p a p e r s and then floated on 2 p. cent u r a n y l f o r m a t e for several minutes). T h e grid w a s then d r i e d by t o u c h i n g one edge to filter paper. The X-ray s c a t t e r i n g m e t h o d s h a v e been prev i o u s l y d e s c r i b e d [5, 6].
I_
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Fro. 1. - - Sketches of ,two nudeohistone models. The Bram and Ris model is a non-uniform coil having an average pitch of 5'0 A but for convenience is drawn here as a regular coil.
loop of I h e 100 A fiber folded u p o n itself. The s t r o n g equatorial reflections at 60 and 30 A w o u l d be e x p e c t e d f r o m the i n t e r p a r t i c l e spacings of m o l e c u l e s h a v i n g a cross-section r a d i u s of gyration of 30 A. T h e 38 and 22 A reflections w o u l d then resnlt f r o m the average p a r a m e t e r s of the ind i v i d u a l particles. Spacings at 40 and 23 A are o b s e r v e d in the X-ray s c a t t e r i n g p a t t e r n of i n d i v i d u a l molecules in solution and gels ; these spacings and t h e i r relative intensities c o r r e s p o n d w e l l w i t h those e x p e c t e d f r o m the m o d e l of Brain and Ris [6].
MATERIAL AND METHODS. N u c l e o h i s t o n e was isolated f r o m calf t h y m u s e m p l o y i n g a m o d i f i c a t i o n of the Z u b a y a n d Doty p r o c e d u r e [9]. The m e t h o d i n v o l v e s the use of m u c h less s h e a r and an isolation pH of 6.5 i n s t e a d of 8.0 [5, 6]. N u c l e o h i s t o n e isolated at p H 8.0 h a d a p o o r l y defined outline in e l e e t r o n m i c r o g r a p h s and e x h i b i t e d m u c h m o r e side by side aggregation than m a t e r i a l isolated at pH 6.5 [51. F o r study in
BIOCHIMIE, 19,72, 54, n ° 8.
T H E NUMBER OF DNAs P E R CROSSECTION T h e d i a m e t e r of n u c l e o h i s t o n e fibers is occasionally o b s e r v e d to be as small as 25 A in stretches contiguous w i t h 80-10,0 A t h i c k r e g i o n s (see fig. 2b). This observation, plus p a r t i a l p r o n a s e digestion studies of n u c l e o h i s t o n e on e l e c t r o n mic r o s c o p e grids [3], r e q u i r e that there be only one DNA double h e l i x p e r cross section, coiled or folded to m a k e a fiber 100 X in diameter. I n c o n t r o vertible e v i d e n c e for a s u p e r coiled s t r u c t u r e is the r e v e r s i b l e a p p e a r a n c e of a well defined DNA X-ray p a t t e r n and the d i s a p p e a r a n c e of the 38-40 X spac i n g u p o n the s t r e t c h i n g of well o r i e n t e d nuc]eoh i s t o n e fibers [4]. SMAEL AND INTE,RM~,DIATE ANGLE X-RAY SCATTERING. The entire n u c l e o h i s t o n e X-ray s c a t t e r i n g pattern w a s f o u n d to be i n v a r i a n t o v e r at least the r a n g e of i o n i c strength b e t w e e n 0.8 and 20 mM s o d i m n p h o s p h a t e at pH 6.8 [5, 6]. F r o m the small angle X-ray s c a t t e r i n g w e h a v e o b t a i n e d a cross section r a d i u s of g y r a t i o n of 30 A and a mass p e r unit length of 12010 Daltons/3~ [6] w h i c h are in r e a s o n a b l y good a g r e e m e n t w i t h the values of 26 A and 150,0 Daltons/3~ o b t a i n e d by Luzzati and Nicolaieff [8]. T h e cross section r a d i u s of gyration Re, is defined as the root m e a n s q u a r e dist a n c e of electrons f r o m the long axis. F o r a structure c o n s t r u c t e d out of one 25 A thread, a R e of 30 A r e q u i r e s that its c e n t e r be at an a v e r a g e of 28 A f r o m the axis. Such a s t r u c t u r e is r a t h e r c o m p a c t as the c e n t e r <
A structural chromosome w i d e (see fig. 1 a). T h e 30 A e x p e r i m e n t a l c r o s s s e c t i o n r a d i u s of g y r a t i o n a g r e e s w e l l w i t h t h e diameter observed in the electron microscope but n o t at all w i t h t h e 50 A c r o s s s e c t i o n r a d i u s o f g y r a t i o n e x p e c t e d f r o m t h e P a r d o n el al. m o d e l .
model.
1007
experimental M/L represents a minimum vahm, f o r if e x t e n s i v e s i d e b y s i d e a g g r e g a t i o n w e r e p r e sent the e x p e r i m e n t a l result w o u l d be less t h a n t h e t r u e v a l u e o f t h e i s o l a t e d p a r t i c l e s . But f r o m t h e m i n i m u m v a l u e of t h e m a s s p e r u n i t l e n g t h
FifunE 2; a, b, and e are thin sections of an embedded nucleohistone pellet positively stained w i t h lead citrate and uranyl acetate. FI6. 2 a. - - <
The mass per unit length contains information about the d i s t r i b u t i o n of mass in the d i r e c t i o n of l h e l o n g axis. T h e m a s s p e r u n i t l e n g t h c o n t r i b u t i o n of t h e DNA i n n u c l e o h i s t o n e is 3-4 t i m e s t h a t of i s o l a t e d B-DNA (18,6 D a l t o n s / A ) ; t h e f a c t t h a t t h e r e is o n l y o n e DNA p e r c r o s s s e c t i o n r e q u i r e s t h a t t h e DNA b e f o l d e d o r s u p e r c o i l e d to give a 3-4 f o l d c o n t r a c t i o n f r o m its e x t e n d e d l e n g t h . T h e
BIOCHIMIE, 1972, 54, n ° 8.
one can calculate a m a x i m u m value of the s u p e r coil p i t c h . Ii c a n b e s h o w n t h a t t h e p i i c h m u s t be s m a l l e r t h a n a b o u t 60 A, a n d a v a l u e o f 50 A w o u l d be in better a g r e e m e n t w i t h the results
~5, 6j. Independent information concerning the shape of l h e n u c l e o h i s t o n e p a r t i c l e is o b t a i n a b l e f r o m t h e X - r a y s c a t t e r i n g p a t t e r n at i n t e r m e d i a t e s c a t -
Stanley Brain.
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t e r i n g angles. In this region of the scattering p a t t e r n the shape of the curve is a f u n c t i o n of the shape of the particle. Two m a x i m a in the plot of the i n t e n s i t y scattered vs, scattering angle are observed, one at an equivalent bragg s p a c i n g of 4.0 A and the other at 22 A. W h e n the scattering p a t t e r n is c o m p a r e d to curves calculated for various type of models, coils were f o u n d to give the best agreement [5~. W i t h a super coil model h a v i n g an R e of 3,0 A and a p i t c h of 45 A an exact fit in both the s p a c i n g a n d relative intensities of the e x p e r i m e n t a l curve was obtained. However, the e x p e r i m e n t a l m a x i m a were m u c h b r o a d e r t h a n those calculated for a regular helix. This implies that the coiling is not regular. W h e n nlodel structures featuring a v a r i a t i o n i n r a d i u s a n d pitch were employed a good fit to the X-ray scattering was o b t a i n e d [5]. ELECTRON MICROSCOPY. My m a i n objective i n p r e s e n t i n g these micrographs is to show that the t e r t i a r y s t r u c t u r e of n u c l e o h i s t o n e is compact, a n d i m p e n e t r a b l e to p r o t e i n molecules. For example, a small p r o t e i n such as lysozyme has a diameter of 50 A and the lac repressor w h i c h is active as a t e t r a m e r [12] w o u l d have a m u c h larger diameter. I suggest that the r e a d e r i m a g i n e a 6,0 A sphere (which is about 2/3 the diameter of the fibers) and ask w h e t h e r or not this sphere w o u l d p e n e t r a t e into the i n t e r i o r of the images. By this c r i t e r i a the model of Brain a n d Ris is a very compact s t r u c t u r e - - a sphere 30 A in diameter w o u l d not p e n e t r a t e the gyres of the super coil. I n fact, the spaces b e t w e e n the gyres of this model are sufficiently small to allow for the histones of n e i g h b o r i n g gyres to form h y d r o p h o b i c linkages and to t h e r e b y stabilize the conformation. Figure 2 shows high resolution electron micrographs of calf t h y m u s n u c l e o h i s t o n e e m b e d d e d in t h i n epoxy sections. The n u c l e o h i s t o n e has the same general a p p e a r a n c e as in sections of whole nuclei [13]. This n u c l e o h i s t o n e [6] also r e t a i n s the m o r p h o l o g y of c h r o m a t i n fibrils o b t a i n e d by s p r e a d i n g open whole cells on an air salt solution i n t e r p h a s e [3, 6~. The 10,0 A fibers are seen to be c o n s t r u c t e d out of a t h i n n e r fiber 20-30 A i n diameter w h i c h folds up to make the t h i c k e r fiber. At times the folding can be r e a d i l y followed (see figure 2 a a n d c). Bands of contrast r u n n i n g at large angles to the axis are often observed ; they sometimes have the a p p e a r a n c e of gyres i n a coil. The distance b e t w e e n these b a n d s is 40-50 A, w h i c h is in agreement w i t h the X-ray results. However, the structure is usually not r e g u l a r - -
BIOCHIMIE, 19,72, 54, n ° 8.
that is, some regions are more folded t h a n others. But the most i m p o r t a n t observation is that the u l t r a s t r u c t u r e appears to be quite compact. Very few regions are seen w h e r e a 60 A sphere could penetrate, a n d i n general the i n t e r i o r of the substructure appears to be inaccessible to molecules larger t h a n 30 to 40 A. Such a c o m p a c t folding was also observed b y Wettstein a n d Sotelo i n their sections of n u c l e i [13]. I n figure 2 b a rare stretch of u n f o l d e d chromatin is noticed. These u n f o l d e d regions make up at most a few p e r c e n t of the total mass, w h i c h disagrees w i t h the extensive u n f o l d i n g p r o p o s e d by Clark and Felsenfeld [14]. Nucleohistone was also s t u d i e d by negative staining. U n f o r t u n a t e l y , m u c h of the u l t r a s t r u c t u r e is b l u r r e d by the i m p a r t i a l p e n e t r a t i o n of the particles of the u r a n y l formate p r e c i p i t a t e w h i c h are about 15 to 25 A i n diameter. Such a compact structure w h i c h i m p e d e s the p e n e t r a t i o n of these small particles should limit the access of p r o t e i n molecules to the DNA. These e l e c t r o n m i c r o g r a p h s are not at all consistent with the loose super helix of P a r d o n et al. [4] but s u p p o r t the compact structure of the Brain and Bis model.
Wide angle X-rag scattering. The difference between the s e c o n d a r y structure of DNA in solution and in n u c l e o h i s t o n e could be the reason for the t e r t i a r y super coiling of the DNA. Wide angle X-ray scattering e x p e r i m e n t s [15] show that the t u r n angle per base p a i r of the DNA in n u c l e o h i s t o n e is about 10 p. cent larger t h a n i n solution. That is if one assumes that DNA in solution has 10 base p a i r s per t u r n , there are about 9 base pairs p e r repeat i n nucleohistone. If the ends of a DNA region were held fixed, a change in the s e c o n d a r y w i n d i n g w o u l d cause t e r t i a r y s u p e r coiling i n complete analogy to closed c i r c u l a r DNA [15]. F i x a t i o n could be achieved b y p r o t e i n cross links or m u l t i p l e b i n d i n g to the n u c l e a r m e m b r a n e . The i n c r e a s e d right h a n d e d w i n d i n g of a DNA region i n n u c l e o h i s t o n e w o u l d result i n a left h a n d e d super coiling p r o p o r t i o n a l to the change i n w i n d i n g . It has been s h o w n that the 10 p. cent difference b e t w e e n the t u r n angle of DNA i n solution a n d of ~be b u l k DNA in nuclcohistone is consistent w i t h the compact s u p e r coil of tile Bram and Ris model [15]. Thus, the cross seclion r a d i u s of g y r a t i o n a n d mass per u n i t length, i n t e r m e d i a t e angle scattering maxima, the wide angle-X-Ray p a t t e r n a n d elect r o n m i c r o s c o p y all i n d e p e n d e n t l y agree w i t h a compactly folded strncture h a v i n g an average p i t c h of 50 A.
A structural chromosome
RECOGNITION DNA. The results also show that the super coiling is n o n - u n i f o r m . This is a c o n c l u s i o n w h i c h must also be d r a w n from the fact that the various histone fractions differ i n their ability to super coil DNA. F o r example, r e m o v i n g very lysine rich histonc I from n u c l e o h i s t o n e or a d d i n g it to DNA causes no change in the t e r t i a r y structure [16], but the a d d i t i o n of a r g i n i n e r i c h histone results i n the super coiling of the DNA [17]. I have previously s h o w n by w i d e angle scattering experiments that DNA very r i c h in AT base pairs has a s e c o n d a r y structure different from DNA of lower AT content, and have proposed that very AT r i c h DNA serves as m a r k e r s for control a n d recognition [18]. Since h i s t o n e I b i n d s p r e f e r e n t i a l l y to DNA w h i c h is r i c h e r in AT [19], this type of DNA might be kept relatively u n f o l d e d due to the weak s u p e r coiling p o w e r of this histone. Its u n i q u e s e c o n d a r y structure m i g h t also p r e v e n t if from f o r m i n g a tight super coil. I p r o p e s e that - - except for a small a m o u n t of DNA serving a r e c o g n i t i o n role - - the DNA i n chromosomes is tightly folded a n d therefore inaccessible to r e c o g n i t i o n or translation proteins. This m e c h a n i s m w o u l d facilitate the f i n d i n g of a correct r e c o g n i t i o n site in the chromosomal mass, as only a small p o r t i o n of the DNA could be sampled. Crick has p r o p o s e d that the DNA used for recognition is tightly folded, and that w h i c h codes for p r o t e i n is extended [2~)]. The model proposed here is i n some ways the o p p o s i t e : r e c o g n i t i o n DNA represents a m i n o r fraction a n d is relatively extended w h i l e coding DNA r e m a i n s tightly folded (and at the same time o v e r t u r n e d ) unless it is activated by a l o o s e n i n g of the t e r t i a r y and secondary structure. F o r the model as proposed, it is not necessary to c o m m e n t on the relative a m o u n t s of c o d i n g to control ])NA. P h y s i c a l chemical studies of inactive chromatin i m p l y that most, if not all, of the c o n s t i t u e n t DNA is double s t r a n d e d [4, 6, 9, 15] a n d i n i t i a l r e c o g n i t i o n would be expected to be of double s t r a n d e d DNA as is the case for the lac a n d phage repressor [21, 22]. Specificity could be achieved by two or three short b i n d i n g regions of different s e c o n d a r y structure separated by a requisite n u m b e r of base pairs. But it is more reasonable to suggest that r e c o g n i t i o n involves a two step process : first the semi-specific b i n d i n g to a general type of DNA, followed b y a r e c o g n i z e r induced modification of the s e c o n d a r y structure. Only the correct D~NA site will be distorted so as to give a u n i q u e strong b i n d i n g configuration with BIOCHIMIE, 1972, 54, n ° 8.
1009
model.
a p r o d u c t i v e fit. I have p r e v i o u s l y suggested that these general r e c o g n i t i o n regions are very r i c h i n AT [151 ; c o n s e q u e n t l y very AT r i c h DNA could competed for recognizers by n o n - p r o d u c t i v e binding as has been s h o w n for the lac repressor [see reference 30]. To be t r a n s c r i b e d coding DNA must u n w i n d , at least partialy, w h i c h w o u l d r e q u i r e an equivalent w i n d i n g of the n e i g h b o r i n g DNA. The adjacent DNA is c o n s t r a i n e d from f u r t h e r w i n d i n g by its histone i n d u c e d compact s e c o n d a r y structure ; however, d e n a t u r a t i o n or over w i n d i n g of "the adjacent r e c o g n i t i o n site w o u l d remove this const r a i n t a n d the coding DNA could u n w i n d for transcription.
P R O P E R T I E S OF VERY AT RICH DNA. lit is p e r t i n e n t to c o n s i d e r w h a t we k n o w about the s e c o n d a r y structure of very AT r i c h DNA. Figure 3 shows the wide angle s c a t t e r i n g p a t t e r n s of a series of I)NAs of different base c o m p o s i t i o n in .05 NaCl. Up to about 60 p. cent AT the wide
~
~ :
6,0 0,10
0,15
0,20
$¢atterin 9 angle 15,4
10,3
0,25
' ) 0,30
6,2
5,2
radiorts
7,7 o Sp|A)
FIG. 3. The wide angle scattering patterns for solutions of DNA from E. colt (E):, M. lysodeikiticus (M), calf .thymus (T), Cl. Perfringens (C) and B. cereus (B) in. 05M NaC1. (After reference [181). -
-
angle p a t t e r n s are similar, but at about 65 p. cent AT (A + T / G + C : 2.0) a significant change occurs ; all of the m a x i m a are c o n s i d e r a b l y flattened, especially the one near 13 A, a n d the maxim u m at 10 A is significantly i n c r e a s e d in i n t e n s i t y c o m p a r e d to the first. Thus the s e c o n d a r y structure must be different from that of DNA of typical base c o n t e n t i n solution. Recent X-ray fiber
Stanley Brain.
1010
d i f f r a c t i o n e x p e r i m e n t s s h o w that v e r y AT r i c h DNA in fibers adopts several n e w f o r m s w h i c h are not s h o w n by t y p i c a l DNA [23, 24]. T h i s polym o r p h i s m is consistant w i t h the r e c o g n i t i o n function p r o p o s e d for AT r i c h DNA. On the basis of some r a t h e r scanty X-ray data for AT r i c h DNA a n d f r o m s t e r i o c h e m i c a l cons i d e r a t i o n s I suggested that v e r y AT r i c h DNA m i g h t not a d o p t the A f o r m [18], and s u b s e q u e n t X-ray fiber d i f f r a c t i o n studies in o u r l a b o r a t o r y s u p p o r t this suggestion [24], DNA that is r i c h e r t h a n about 65 p. cent AT has not been f o u n d to adopt the A form, u n d e r c o n d i t i o n s w h i c h give this c o n f o r m a t i o n for DNA less r i c h in AT. This
is so for those that have been c h a r a c t e r i s e d in procaryotes : the lac o p e r a t o r [30] - - and)~ prom o t o r [31] (*). It w o u l d be in a g r e e m e n t w i t h the m o d e l if c y t o p l a s m i c RNA f r o m h i g h e r cells w e r e of l o w e r AT c o n t e n t t h a n the bulk DNA. I n d e e d , this seems the case for those R NAs w h o s e base contents h a v e been d e t e r m i n e d (see table I). T h e h i g h GC c o n t e n t of h i s t o n e m e s s e n g e r is p a r t i c u l a r l y n o t e w o r t h y , for h i s t o n e c o n t a i n s large amounts of l y i s n e w h i c h is c o d e d f o r by e i t h e r AAA or AAG, both of w h i c h substantially i n c r e a s e the AU c o n t e n t of the m e s s e n g e r ; the messenger, h o w e v e r is GC rich. T h e h i g h (~C c o n t e n t of these c y t o p l a s m i c RNAs could also be due to the prefer e n t i a l f o r m a t i o n of double s t r a n d e d r e g i o n s
TABLE I.
Base compositions of cytoplasmic RNAs of higher organisms (1). A+U
RNA
Calf eye lens messenger (32) . . . . . . . . . . . . . . . . . . . . . . Hemoglobin messenger (33) . . . . . . . . . . . . . . . . . . . . . . . Calf histone messenger (34) . . . . . . . . . . . . . . . . . . . . . . . Xenopus (35) 5 S RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transfer HNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 S ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 S ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bulk DNA
A+T G+C
0,97 0,71 0,88 1.3tol.4
0,75 0,67 0,85 0,59
(1) The possible presence, of poly A in the messengers has not. been accounted for, however it could only rncrease the GC content of the remainder. is also i n f e r r e d f r o m i n f r a r e d studies [251. (it is w o r t h m e n t i o n i n g h e r e that although p o l y [d(A-T)] • p o l y [d(A-T)] does a d o p t the A f o r m [263, p o l y (dA) • p o l y (dT) does not [27]).The i d e a that the B-A t r a n s i t i o n is i n v o l v e d in t r a n s c r i p t i o n w a s p r o p o s e d by c r y s t a l l o g r a p h e r s because the RNA-DNA h y b r i d is f o u n d only in the A-form [28]. T h a t t r a n s c r i p t i o n r e q u i r e s a B-A t r a n s i t i o n is strongly i m p l i e d f r o m r e c e n t studies s h o w i n g that the c i r c u l a r d i c h r o i s m s p e c t r u m of the RNA polym e r a s e - s i g m a b i n d i n g site goes f r o m the B to the A t y p e s p e c t r u m u p o n starting RNA synthesis [29]. If a B to A t r a n s i t i o n must p r e c e d e t r a n s c r i p t i o n , v e r y AT r i c h DNA in h i g h e r o r g a n i s m s m i g h t not be t r a n s c r i b e d w i t h the usual t r a n s l a t i o n system d e s i g n e d for t y p i c a l DNA b e c a u s e it does not adopt e i t h e r the B or A form.
which would attack.
be
resistent
to
certain
nuclease
It w o u l d be w o r t h w i l e in studies of s p a c e r and satellile DNAs to i n v e s t i g a t e w h e t h e r or not the g e n e t i c code has a r e s t r i c t e d r a n g e of a l l o w a b l e base contents and sequences. In a d d i t i o n to the a d v a n t a g e of setting aside classes of DNA to serve roles o t h e r than coding, these c r i t e r i a w o u l d provide a m e a n s of l i m i t i n g the a p p e a r a n c e in m-RNA of the n o n c o d i n g t e r m i n a t i o n codons UAG and UAA w h i c h are m o r e than 66 p. cent AU.
Acknolwledgements. I thank David Perrin for his interest and many helpful suggestions. These ideas were presented at the Colloquium of the Socidt6 Chimie Biologique in Strasbourg, March 1972.
SOME P R E D I C T I O N S OF T H E MODI~L. The m o d e l p r e d i c t s that o p e r a t o r and p r o m o t e r sites s h o u l d be in v e r y AT r i c h regions, a n d this
BIOCHIMIE, 1972, 54, n ° 8.
(*) I't has recently been found tha,t the RNA polymerase binding sites of phage fd replicative form DNA are very AT rich and are also double stranded [36].
A s t r u c t u r a l c h r o m o s o m e model. RfiscM~. Le mod61e p r o p o s 6 p e u t ~tre r 6 s u m 6 c o m m e s u i t : 1) La p l u s g r a n d e p a r t i e de t ' A D N d a n s les c h r o m o s o m e s e s t n o n u n i f o r m 6 m e n t r e p l i 6 e en u n e n r o u i c m e n t t e r t i a i r e c o m p a c t g a u c h e d o n t le p a s m o y e n et le r a y o n s o n t de 45 .~. P o u r ce q u i e s t de la s t r u c t u r e s e c o n d a i r e le pas, o u e n c o r e ]e r a y o n de cet ADN e s t de 10 p. c e n t p l u s p e t i t q u ' e n s o l u t i o n . 2) E n v e r t u de cette s t r u c t u r e s e c o n d a i r e r e n v e r s 6 e , la m a j e n r e f r a c t i o n de cot ADN n e p e u t 6tre t r a d u i t . C o n c u r r e m m e n t , la s t r u c t u r e t e r t i a i r e c o m p a c t e b l o q u e ~l'acc6s d e s m o l 6 c u l e s de r e c o n n a i s s a n c e et d e p o l y m 6 r a s e h c~t ADN. 3) Seule u n e p e t i t e f r a c t i o n de I'ADN t o t a l n ' e s t p a s 6 t r o i t e m e n t repli4e ct e s t p a r c o n s 6 q u e n t u t i l i s a b l e p o u r la r e c o n n a i s s a n c e . I1 est s u g g 6 r 6 q u e cet ADN de r e c o n n a i s s a n c e a u n e c o m p o s i t i o n en b a s e s n o n t y p i q u e et q u ' e l l e est tr6s r i c h e en AT. L ' A D N de r e c o n n a i s s a n c e est c o n s e r v 6 n o n r e p l i 4 e p a r sa s t r u c t u r e s e c o n d a i r e u n i q u e on s o n i n t e r a c t i o n p r 6 f 6 r e n t i e l l e avec les h i s t o n e s I. La r e c o n n a i s s a n c e spScifique p e u t i m p l i q u e r la l i a i s o n h u n t y p e g 6 n 6 r a l d ' A D N (tr6s r i c h e en AT) s u i v i e p a r n n e t r a n s i t i o n de s t r u c t u r e i n d u i t e p a r la mo16cnle p o u r p e r m e t t r e la r e c o n n a i s s a n c e e x a c t e de la s 6 q n e n c e p r i m a i r e . 5) I1 est prop.os6 q u e d a n s ]a cellule, I'ADN tr6s r i c h e e n AT ne p e u t c o m m e n c e r la t r a n s c r i p t i o n de p a r sa s t r u c t u r e s c c o n d a i r e r e m a r q u a b l e et p a r c e q u ' i l n ' a d o p t e p a s la f o r m e A.
ZUSAMMENFASSUNG. D a s v o r g e s c h l a g e n e Modell k a n n w i e f o l g t z u s a m m c n g e f a s s t w e r d e n : 1. D e r g r 6 s s t e Tell d e r DNS in d e n C h r o m o s o m c n w i r d u n g l e i c h m i i s s i g in e i n e r k o m p a k t e n tertHiren linken Struktur, dcren mittlerer Windungsa b s t a n d u n d R a d i u s 45 .~ be,t~iigt, z u s a m m e n g e f a l t e t . Bet d e r s c k u n d i i r e n S ~ r u k t u r is.t die W i n d u n g s H i n g e o d e r a u c h d e r R a d i u s u m 10' p. c e n t k l e i n e r a l s in LSsung. 2)Wegen dieser umgekehrten sekundiiren Struktur k a n n d i e g r S s s t e F r a k t i o n d i e s e r DNS n i c h t u m g e s e t z t w e r d e n . Z u s a m m e n w i r k e n d v c r h i n d e r t die k o m p a k t e tertiiire Struktur den Zutritt der Rekognoszierungsm o l e k i i l e u n d d e r P o l y m e r a s e z u d i c s e r DNS. 3. A l l e i n e i n c g e r i n g e F r a k t i o n d e r g e s a m t e n DNS w i r d n i c h t e n g g e f a l t e t u n d ist f o l g l i c h fiir d i e R e k o gnoszierung verwendbar. Es wird angenommen, dass diese Rekognoszierung-DNS sich aus nicht-lypischen B a s e n z u s a m m e n s e t z t u n d d a s s sic r e i c h a n AT ist. Die R c k o g n o s z i e r n n g s - D ' N S f a l t e t sich n i c h t d u r c h i h r e b l o s s e sekund~ire S t r u k t u r o d e r d u r c h i h r c v o r z u g s w c i s e W e e h s e B v i r k u n g m i t d e n H i s t o n e n . I. Die s p e z i fische R e k o g n o s z i e r u n g k a n n die B i n d u n g a n e i n e m a l l g e m e i n e n , a n AT s e h r r e i c h e n T y p d c r DNS, die y o n e t h e r S t r u k t u r i i n d c r u n g , die d u r c h d a s Molekiil v o r g e n o m m e n w i r d , u m die g e n a u e R e k o g n o s z i e r u n g der primiiren Sequenz zu erlauben, implizieren. 4. Es w i r d a n g e n o m m e n , d a s s i n de.r ZeHe d i e a n AT s e h r r.eich.e DNS die T r a n s k r i p t i o n a u f G r u n d i h r e r
BIOCHIMIE, 1972, 54, n ° 8.
1011
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Chemical
Basis