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Home , Histone octamer, Nucleosome

Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

Studies of Structure

T.J. RICHMOND, T. RECHSTEINER, AND K. LUGER Institut fur Molekularbiologie und Biophysik, ETH-Honggerberg, CH-8093 Zurich, Switzerland

The nucleosome is the universal /DNA ele- terminal regions in the core particle can be identified. ment repeated throughout the of eukaryotic For example, the entry point for the H4 amino- cells (Kornberg 1977; McGhee and Felsenfeld 1980). terminal "tail" and perhaps the tail itself can be recog- Presumably as a consequence of its fundamental role in nized. The H2A carboxy-terminal and the H3 amino- the organization of DNA in a compact unit, it has also terminal tails appear to form domains adjacent to the been found to take part in regulation (see, e.g., termini of the DNA . The positions of the Pederson et al. 1986; van Holde 1988; Grunstein 1990; histone protein/DNA cross-links are highly consistent Wolffe 1991; Kornberg and Lorch 1992). A major with the new electron density assignments, objective of our laboratory has been to obtain structur- To obtain greater structural detail for the nucleo- al information at high resolution for the nucleosome, its some, crystals of a nucleosome core particle containing higher-order structure, and its interactions with other DNA with a single sequence were prepared and found nuclear factors. Previously, the structure of the nucleo- to diffract to approximately 3.5 ,~ resolution (Rich- some core particle was solved by X-ray crystallography mond et al. 1988). To aid the selection of heavy-atom at 7 A_ resolution using the method of multiple iso- derivatives for structure determination and to permit morphous replacement (Richmond et al. 1984). This mutagenic analysis of particle stability and assembly, X-ray structure confirmed the size and shape of the, we have prepared and crystallized core particles that particle deduced earlier from several lines of infor- contain all the as recombinant . The mation (Finch et al. 1979), showing that the "146"-bp higher-resolution structure should allow accurate DNA is arranged in 1.8 superhelical turns around the identification of the DNA path and location of many of in a disk-like structure 57 A in height the side chains of the histone proteins. Finally, a along the superhelix axis and approximately 110 A, in nucleosome particle that contains 179 bp of DNA and diameter. The DNA was seen to be bent tightly at the globular domain of is being character- several positions along its superhelical path. The shape ized and has been crystallized. and dimensions of the histone octamer within the nucleosome core particle were observed to match those Preparation of Nucleosome Core Particle Crystals measured from the octamer structure at 20 A, resolution determined by image reconstruction from electron mi- Nucleosome core particles can be obtained by lysis of crographs made from ordered aggregates under high- cell nuclei and partial digestion of the exposed chro- salt conditions (Klug et al. 1980). Even at low res- matin with micrococcal nuclease. The particles contain olution, the DNA superhelix was seen to follow a approximately 147 -+ 2 base pairs of DNA, representing helical ramp formed by the histone proteins. Regions of the natural abundance of DNA sequences (i.e., con- the histone octamer were assigned to the four pairs of taining mixed sequences), and two copies of each of the histones, H3, H4, H2A, and H2B, first in the 20 A, four core histones, H2A, H2B, H3, and H4, for a resolution structure, and then in the 7 A, X-ray struc- roughly equal mass of protein and DNA (Finch et al, ture, based on the DMS method of protein/DNA 1977). These particles of 206 kD yield crystals that cross-linking (Mirzabekov et al. 1978; Shick et al. diffract anisotropically to 3.5-7 A~ resolution and have 1980). Although many internal features of the histones resulted in a structure at 7 A, resolution (Finch et al. were seen, their assignment to individual histones could 1979; Richmond et al. 1984; Rhodes et al. 1989). be made only rather crudely, since the polypeptide To improve the X-ray diffraction from nucleosome chain could not be identified by recognition of indi- core particle crystals, the mixed sequence DNA was vidual side chains. replaced with a 146-bp DNA fragment of unique se- In light of the reinterpretation of the histone octamer quence (Richmond et al. 1988). The sequence selected crystal structure (Arents et al, 1991), it is now possible was from the 5S RNA gene of Lytechinus variegatus to assign the location of the histone proteins in the and contains the distal 25 bp of the factor nucleosome core particle structure more accurately. IIIA (TFIIIA) binding site (Simpson and Stafford The location of essentially all the c~-helical regions 1983). After insertion of this fragment in multiple identified from the histone octamer crystals can be copies in a , amplification in bacteria, restric- located in the nucleosome core particle X-ray structure. tion enzyme excision, and high-performance liquid Although some rearrangement of structural elements is chromatography (HPLC) purification, core particles apparent, the entry or exit points of most of the histone were reconstituted in high yields using histone octamer

Cold Spring Harbor Symposia on Quantltatwe Btology, Volume LVIII. 91993 Cold Spring Harbor Laboratory Press 0-87969-065-8/93 $5.00 265 Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

266 RICHMOND, RECHSTEINER, AND LUGER

from chicken erythrocytes. Crystals of these particles measuring along several turns of an c~ helix. Since the diffract anisotropically, beyond 3 A in the best direc- molecular (also crystallographic) twofold axis in the tion and to 3.5-4 A in the poorest direction. Compared histone octamer is along the horizontal in Figure 3c, it to the mixed sequence crystals which have one particle must also be close to horizontal in Figure 5. Given the in the crystallographic asymmetric unit (asu), these lack of perfect mirror symmetry about a horizontal line defined sequence crystals have two particles for 412 kD for the outline of the whole octamer in Figure 3, the per asu. twofold axis must be somewhat out of the plane of the More recently, totally recombinant nucleosome core projection. It cannot lie very far out, however, since it particles have been produced using histone from must be perpendicular to the molecular superhelix axis, Xenopus laevis (Old et al. 1982) expressed in bacteria and the view in Figure 5, as noted by the authors, is (Studier et al. 1990; K. Luger et al., in prep.). Various approximately down the superhelix axis. By including a mutant histones have been prepared, and nucleosome second transparent copy of the Figure 5 ribbon diagram core particle crystals containing them have been grown. inverted about a horizontal axis in the previous Fragments of defined sequence DNA longer than the superposition, both ribbon diagrams can be translated 146 bp have also been made and reconstituted with the as required to fit the surface projections of the "pseu- do" space-filling model shown in Figure 3c. The core histones into particles which crystallize. In addi- twofold axis position determined in this manner relat- tion, the globular domain of histone H1 (Hlg), the ing the half octamers in the ribbon diagrams falls on the outer or linker histone protein, from X. laevis has also most obvious location for the twofold axis in the surface been expressed in bacteria. Hlg has been used to representation. assemble and crystallize a nucleosome containing all The tetrakismercurimethane compound (TAMM) five histone proteins and 179 bp of DNA (T. Rech- used to make the single heavy-atom derivative for the steiner and T.J. Richmond, in prep.). hsHO structure determination is most probably bound to the Cys-ll0 residues, the only cysteine Assignment of the Histone Proteins in the Nucleosome groups in the molecule. Using coordinates for the Core Particle at 7 TAMM multi-heavy-atom group given by Arents et al. (1991), which apparently corresponds to a single mer- The electron density map of the histone octamer cury atom because of chemical decomposition (see crystals obtained at high salt concentrations has been Burlingame et al. 1985), the site is approximately 1 recalculated (Arents et al. 1991) and found to be away from the molecular dyad axis and adjacent to the substantially different from the map originally deter- carboxyl terminus of the H3 long helix. According to mined (Burlingame et al. 1985). The distribution of the the secondary structure assignments reported in Figure polypeptide chains is substantially altered from the 2 of Arents et al. (1991), H3 Cys-110 is located in the previous interpretation. The original histone octamer carboxy-terminal turn of this long a helix. This site structure had maximal dimensions of 110 A, • 65-70 provides a common structural marker very near the A, which have been reduced to 60 A, x 65 A in the molecular symmetry axis in both the hsHO and msNCP republished structure. These dimensions compare well histone octamer structures. with those for the 20 A resolution histone octamer In the presentation of the hsHS and msNCP histone structure reported as 56 A x 70 ~, (Klug et al. 1980). octamer structures, the views given in Figure 5 of The most recent structure of the "high salt'" histone Arents et al. (1991) and in all figures of Richmond et octamer (hsHO) using data to 3.1 A resolution war- al. (1984), respectively, are views approximately down rants comparison to the histone octamer structure the molecular superhelix axis. By alignment of the dyad observed in the X-ray structure of the mixed sequence axis and heavy-atom site for a scaled, transparent copy nucleosome core particle (msNCP) at 7 A, resolution of the hsHO ribbon diagram onto the msNCP electron (Richmond et al. 1984). Although the atomic coordi- density map, a striking correspondence in location and nates for the hsHO structure are not yet available, orientation of the major structural units is observed. sufficient information has been presented in the repub- The order of appearance of the structural elements lication to permit a comparison of the predominant seen in the core particle map in either of the two features of both the hsHO and msNCP structures opposing views down the superhelix axis agrees in (Arents et al. 1991). detail with that seen in the stereo diagram of the hsHO. The chain traces (ribbon diagram representation of On the basis of this comparison, the histone assign- the main chain paths) of one of each type of histone ments for the nucleosome core particle electron density protein for the hsHO are presented in Figure 5 of map made by Richmond et al. (1984) require revision. Arents et al. (1991). By overlaying a transparent copy The major reassignments of the electron density for the of Figure 5 on Figure 3c of Arents et al. (1991), it was histone proteins can be summarized approximately as noted that without relative rotation of the pictures, the follows: surface structural features are well aligned, The out- lines of the molecular images are matched, and the 1. The former H3 region is divided between the H3 superpositions of the ends of the long helices in H4 and and H4 molecules. H2A and the carboxy-terminal helix of H2B are clear. 2. The former H4 domains become predominantly the The scale of one image can be adjusted to the other by H2B molecules. Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

NUCLEOSOME STRUCTURE 267

3. The former H2B domains are divided between the path of the remaining 63 amino acids of the H3 amino H2A and H2B molecules. terminus is discussed below. 4. The former H2A regions become assemblies of In map sections moving up the superhelix axis, closer histone terminal regions, probably formed from a to the outside of the particle, the long a-helical rod combination of the H3 amino termini and the H2A (Fig. 2, H4: 50-74) of the H42 molecule (Fig. lb, green carboxyl termini. rod labeled H42) is seen aligned nearly parallel to the 5. Several polypeptide loops connecting a helices are molecular dyad axis. The polypeptide loop connecting now assigned to follow the phosphodiester back- this helix to the amino-terminal helix of H42 (Fig. lb, bone on the inside of the particle using density labeled N) interacts with the phosphate chain facing previously assigned to the DNA. inward at position 0 2/3. This H4 loop is running in the opposite direction with respect to the adjacent loop of The new interpretation is shown in Figure 1, where H3 interacting with the DNA at position 0 1/3. The all views are approximately down the molecular super- other copy of the long a helix of H4 ~ is seen in the view helix axis. Each copy of each histone molecule contains from the opposite side of the particle (Fig. lc, green a long a helix that is easily identified in the nucleosome rod labeled H41). In this case, the sections extend to core particle map. From the core particle map, it the outside of the particle, and the amino-terminal appears that the polypeptide chain extending from both region of H41 (Fig. lc, labeled T) is seen extending ends of all 8 long a helices interact with a different from the amino-terminal helix (Fig. lc, labeled N on region of the phosphodiester chain on the inside of the green density). This density appears to contact the particle. This interpretation suggests that histone poly- carboxyl terminus of H2B: extending from an adjacent peptide loops make contact with 16 of the 24 possible core particle. Protein/DNA cross-linking data indicate inward-facing stretches of the phosphodiester chains. that this terminus can extend at least as far as to DNA Of the 16 a helices immediately preceding or following position -3 in the isolated core particle (Pruss and these loops, 8 are in association with the DNA back- Wolfe 1993). The loop of polypeptide extending from bone. These histone protein/DNA associations are the carboxyl terminus of the long a helix of H41 follows listed in Table 1, where the DNA backbone positions the phosphate chain at position -2 2/3, perhaps with facing directly inward toward the DNA superhelix axis interactions into the neighboring minor groove, and are labeled as the number of double helix turns from connects to the carboxy-terminal helix below (Fig. 2, the center of the DNA fragment. In accord with this H4: 84-93). This carboxy-terminal helix is seen clearly description, positions ...-2, -1, 0, +1, +2... are for H42 as well (Fig. lb), but the loop density is where the minor groove faces outward, and positions incomplete at the selected level of contouring. In each ... - 2.5, -1.5, +1.5, +2.5... are where it faces inward. half of the H3H4 tetramer, the single long helix in each The molecular dyad axis intersects the double helix at of H3 and H4 interact and cross over each other at an position 0. Also included in Table 1 are the identities of angle of approximately 40 ~. The helical rods and con- the histone proteins yielding the major products in the necting loops of the H3H4 tetramer organize the 6 neighborhood of each position based on protein/DNA central turns of the DNA double helix from positions cross-linking studies (Belyavsky et al. 1980; Bavykin et -3 to +3. al. 1985; Ebralidse et al. 1988). The cross-links iden- The long a helices of H2A and H2B in each tified are all within 1/3 double helical turn of a contact H2AH2B dimer associate in the manner described for by the corresponding histone type, with the exception the pairs of long helices in the H3H4 tetramer. The of the cross-links 0 1/3-H2A and 6 1/3-H3. These two long a helix (Fig. 2, H2B: 56-84) ofH2B 2 (Fig. lb, red apparent violations can be explained by the probable rod labeled H2B2) and of H2B~ (Fig. lc, underlying locations of two histone tail regions. A detailed descrip- red rod) are seen with carboxy-terminal extensions tion of the nucleosome core particle histone assign- looping across the minor grooves at positions 3 1/2 and ments follows. -3 1/2, respectively. These loops follow the phosphate The H3H4 tetramer can be divided into two halves chain on the inside of the particle and lead to two related by the molecular twofold axis of the histone carboxy-terminal helices that are folded back on each octamer. The long a helices (Fig. 2, H3: 87-112) of other (Fig. lc, labeled H2B~). In further sections of the both H3 molecules (Fig. la, blue rods labeled H31 and map, they extend to adjacent particles and appear to H32) make a V shape, as seen in the sections of the interact with the amino-terminal tails of H4 molecules electron density map representing the center of the as noted above. These proposed interparticle interac- particle. The peptide loops that connect these helices to tions may have interesting consequences for nucleo- their respective carboxy-terminal helices (Fig. la, some higher-order structure, or alternatively, they may labeled C for H32) follow the phosphate chains at be an artifact of crystallization. The H2B amino-termi- position 0 1/3. The protein chain extending from the nal helix (Fig. 2, H2B: 38-47), according to the hsHO amino terminus of the long helix of H32 follows the structure, is directed into the structure from the outside phosphate chain at position 2 1/3 to the surface of the of the particle. It appears to lie in the major groove particle, turns, and reenters the particle as the penulti- facing inward at DNA position -+5 (Fig. lc, labeled N mate amino-terminal a helix (Fig. 2, H3: 64-75) inter- on red density at DNA position -5). The long loop acting with the phosphate chain at position 1 2/3. The connecting this helix and the long a helix runs along the Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

268 RICHMOND, RECHSTEINER, AND LUGER

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NUCLEOSOME STRUCTURE 269

Table 1. Histone Protein/DNA Contact Regions DNA position a Hmtone protein contact b Histone cross-linkc 0 k H32 loop from C-terminus of long a helix H3, H2A 0~ H4_, loop from N-terminus of long a helix H4 1 1 H4, N-terminus c~ helix H3, H4 (N-terminus) 13 H3~ penultimate N-terminus a helix H4 --39 • H3~ loop from N-terminus of long a helix H3 2 3 H4, loop from C-terminus of long a helix H4 3!3 H213, loop from C-terminus of long a helix H2B 3 3 H2B 2 loop from C-terminus of long a helix H2B 4•3 H2A, N-terminus c~ helix H2B 43 H2B 2 N-terminus c~ helix H2B 589 H2B 2 loop from N-terminus of long c~ hehx H2A

6•3 H2A, loop from C-terminus of long a helix H3 6~ 7 Regions of the hlstoue proteins that contact positions along the DNA superhehx in the nucleosome core particle are identified by comparison of the histone core of the core particle structure (Richmond et al. 1984) with the free histone octamer structure (Arents et al. 1991). Phosphate chain positions facing inward toward the hlstone proteins as measured in turns of the double hehx from its center at position 0. The nl~ and n 3 positions correspond to mdivldual phosphate chains, where n increases In the 5' to 3' direction for the n 3 chain and 3' to 5' for the n13 chain. b Contacts for only 1/2 of the DNA superhehx are listed since the twofold related positions are essentially identical c Data from Mlrzabekov and co-workers (Belyavsky et al. 1980; Bavykln et al 1985, Ebrahdse et al. 1988).

phosphate chain adjacent to the amino-terminal helix. as the H2A central domains (see, e.g., Fig. lc, labeled For both copies of H2B, this helix fades out as it enters U). However, the central regions of all the histones in an open region, probably because it is no longer helical, the msNCP can be accounted for by comparison with and then possibly reappears near the H4 carboxyl the hsHO structure without assignment of these re- terminus. If this interpretation is correct, it would place gions. Three long terminal polypeptides extend from the amino terminus of H2B 1 and the carboxyl terminus the first or last a helix in three of the histone structures: of H42 in juxtaposition and likewise for the H2B2, H4a the amino termini of H2B and H3, and the carboxyl pair. terminus of H2A (Fig. 2). The histone tail regions are As for the other histones, the long a-helical rod of not observed in the hsHO structure, and therefore their H2A 1 (Fig. 2, H2A: 46-74) is connected at its amino identification in the msNCP structure is largely hypo- terminus to a loop which follows along a phosphate thetical. Each carboxy-terminal tail of H2A presumab- chain, in this case, facing inward at site -4 1/3 (Fig. lc, ly constitutes one of these unassigned regions adjacent labeled N on yellow density). The carboxy-terminal to the terminus of the DNA, since the carboxy-terminal loop approaches the phosphate chain near the end of a helix of H2A is adjacent to this density (Fig. lc, the DNA at position -6 1/3 and continues as an a helix labeled C on yellow). As implied by our earlier work, it (Fig. 2, H2A: 80-90) within the particle. may extend to the outer diameter of the particle and be The regions of electron density beginning within the involved in the binding of the 10-bp extensions that DNA superhelix and extending out over its central gyre would be present in a 166-bp particle (Struck et al. on both faces of the particle were formerly designated 1992). As noted above, the penultimate amino-termi-

Figure 1. Electron density of the mixed sequence nucleosome core particle. The nucleosome core particle structure at 7 resolution is shown m views approximately along the DNA superhelix axis. The particle extends from one protein face with a limit at about section 12 to the other at about section -20. The molecular twofold axis lies approximately parallel to the plane of the figure (heavy lines). The first map contour is drawn at 5.5 times the rms deviation of the electron density to provide clarity through multiple sections. The following color scheme is used: (DNA) brown, (H3) blue, (H4) green, (H2A) yellow, (H2B) red. Selected positions or turns of the DNA double helix from the center of the particle are labeled in accordance with Table 1. (a) Central H31H32 region, map sections 2 to -7. The molecular dyad axis lies closest to section -2. The predominant long a helices of H3 a and H3~ are labeled. The H32 carboxy-terminal helix is labeled C. (b) H32H42 tetramer region, map sections 6 to -1. The predominant long a helices of H42 and H2B 2 are labeled. The H4 z amino-terminal helix is labeled N. (c) H2AxH2B 1 dimer region, map sections -17 to -8. This view is in the opposite direction compared to those of a and b. The predominant long a helices of H41 and H2A 1 are labeled. The H41 amino-terminal helix and tail are labeled N and T, respectively. The H2A 1 penultimate amino-terminal helix and the H2BI amino-terminal helix are labeled N on yellow and N on red densities, respectively. The H2A t and H2B 1 amino-terminal helices are labeled N and H2B~, respectively. The unassigned region of density (uncolored) which most likely corresponds to the carboxy-terminal tail of H2A combined with the amino-terminal tail and a helix of H3 is labeled U. Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

270 RICHMOND, RECHSTEINER, AND LUGER

H2A

N-SGRGKQGGKARAKAKS-

RSSRAGLQ~PVGRVHRLLRKGNYAERVGA~APVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLOLAIRND

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N-PEPAKSAPAPKKGSKKAVTKTQKKGDKKRKKSRKESY-

SIYVYKVLKQ~t~PDTG~SSKAMG~MNSFVNDIFERIAGEAsRLAHYNKRS~SREI~TAVRLLL~G~LA~HAVSEGTKAVTKYTSS -K-C H3

N- ARTKQTARKSTGGKAPRKQ LATKAARKSAPATGGVKKPHRYRPG-

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-GRTLYGFGG-C Figure 2. Histone protein a-helix assignments. The sequences of the chicken erythrocyte histone proteins are shown where the approximate positions of a helices are highlighted (Laine et al. 1978; Grandy and Dodgson 1987; Brandt and von Holt 1974; Wang et al. 1985). The helix assignments are adapted from Fig. 2 of Arents et al. (1991). The single, predominant long helices in each histone referred to in Fig. 2 have the approximate boundaries (H2A) 46-74, (H2B) 56-84, (H3) 87-112, (H4) 50-74.

nal c~ helix of H3 appears to direct the 63 amino- Both recombinant octamers and the chicken erythro- terminal amino acids including the amino-terminal a cyte octamer (c-octamer) were reconstituted to nucleo- helix into the particle and into the region of uniden- some core particles using a defined sequence 146-bp tified density. Furthermore, it may be that the amino DNA fragment. The integrity of the particles can be terminus of H3 combines with the carboxyl terminus of demonstrated by nondenaturing H2A in the opposite half of the histone octamer (e.g., (Fig. 3), Although all three octamers show a unique H2A, and H32) to cooperate in forming a well-defined rotational position in DNase I footprints, at least two or structure involved in binding the linker DNA. The three translational positions which are shifted by multi- histone/DNA cross-linking data as listed in Table 1 are ples of 10 bp have been mapped by micrococcal nu- consistent with this interpretation. clease trimming followed by restriction enzyme cleav- age (not shown, but see Dong et al. 1990; Pennings et al. 1991). The three bands observed in the gel electro- phoresis patterns shown represent three different trans- A Recombinant Nucleosome Core Particle and a lational positions. H1 -containing Nucleosome The DNA termini appear to be less strongly associ- For the purpose of generating new heavy-atom de- ated with the core histones in the nucleosome core rivatives for the solution of the X-ray structure of the particle prepared with rg-OCtamer missing the histone defined sequence nucleosome core particle, we have terminal sequences, as indicated by their reduced elec- prepared crystals of the particle containing mutant trophoretic mobility. The Stokes' radius for this par- histones. In addition to facilitating the phase determi- ticle has apparently increased, because the histone tails nation of the X-ray diffraction data, we have also begun no longer stabilize the binding of the outer approxi- to examine the effect of site-directed mutation on the mately 1/2 turns of superhelical DNA in the isolated stability of the core particle. High-level expression of particle. These DNA regions also have an increased the four X. laevis histone proteins was achieved in susceptibility toward micrococcal nuclease cleavage as Escherichia coli. The insoluble proteins were purified compared to particles containing the intact histones under denaturing conditions and refolded to histone (data not shown). octamers consisting either of the four full-length recom- Crystallization of nucleosome core particles contain- binant histone proteins (r-octamer) or of the four ing several defined mutants of r-octamer occurred "-resistant" globular domains (rg-OCtamer). readily under conditions similar to those for the chick- Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

NUCLEOSOME STRUCTURE 271

the binding of Hlg in a quantitative manner (Fig. 3). 146 bp DNA 179 bp DNA This result indicates that the linker DNA extensions are m c r m c :,,H', :,H' held more tightly to the surface of the particle in the presence of Hlg, causing an overall reduction in the Stokes' radius of the complex. In support of this inter- pretation, a 165-bp DNA-fragment-containingparticle, an intermediate during nucleolysis, was found to be protected from micrococcal nuclease digestion at least 30 times more effectively when Hlg is bound. Crystals of the 179-bp particle with and without Hlg have been grown. At present, the quality of diffraction from these crystals should allow determination of the orientation of the 2.5 A structure of the globular domain of a "linker" histone (Ramakrishnan et al. 1993) in the nucleosome. a b

Figure 3. In vitro assembled recombinant nucleosome core ACKNOWLEDGMENT particles and a Hlg-containing nucleosome. particles were electrophoresed through 5% polyacrylamide We thank D. Sargent for help with photography. gels. (a) Nucleosome core particles containing a defined se- quence 146-bp DNA fragment and either chicken erythrocyte histone octamer (lane c) or recombinant histone octamer, REFERENCES both intact (lane r) and with the terminal "tail" sequence regions removed (lane rg), were electrophoresed. (b) Nucleo- Arents, G., R.W. Burlingame, B.-C. Wang, W.E. Love, and some core particles containing a defined sequence 179-bp E.N. Moudrianakis. 1991. Revised histone octamer struc- DNA fragment, chicken erythrocyte histone octamer, 'and ture 3 1 ,~ resolution. Proc. Natl. Acad. Sci. 88: 10148. either 0 (lane c), 0.5 (lane .5x Hlg), or 1 (lane 1 x Hlg) Bavykln, S.G., S.I. Usachenko, A.O. Zalensky, and A.D. molar equivalents of the globular domain of histone H1 were Mirzabekov. 1990. Structure of and organiza- electrophoresed. Marker DNA fragments with sizes 540, 489, tion of internucleosomal DNA in chromatin. J. Mol. Biol. 404, 350 (a), 320 (b), 242,190,147, and 110 bp are shown also 212: 495. (lanes m). Belyavsky, A.V., S.G. Bavykin, E.G. Goguadze, and A.D. Mirzabekov. 1980. Primary organization of nucleosomes containing all five hlstones and DNA 175 and 165 base- en-octamer-derived particles using the 5S RNA gene pairs long. J. Mol. Biol. 139: 519. sequence. Each of these particles contains a cysteine Brandt, W.F. and C. von Holt. 1974. The determination of the replacement of an amino acid at a specific site. After primary structure of histone F3 from chicken erythrocytes addition of TAMM or methylmercury, these crystals by automatic Edman degradation. Eur. J. Biochem. should provide suitable heavy-atom derivative data sets 46: 419. Burlingame, R.W.,W.E. Love, B.-C. Wang, R. Hamhn, N.-H. to improve the resolution of the structure determi- Xuong, and E.N. Moudrianakis. 1985. Crystallographic nation. Mapping of sites in projection is currently in structure of the octameric histone core of the nucleosome progress in our laboratory, and full three-dimensional at a resolution of 3.3 ,~. Science 228: 546. data sets will be obtained using intense synchrotron Dong, F., J.C. Hansen, and K.E. van Holde. 1990. DNA and protein determinants of nucleosome positioning on sea radiation. urchin 5S rRNA gene sequences in vitro. Proc. Natl. Acad. With the aim of understanding the association of the Sci. 87: 5724. histone H1 within the nucleosome, and eventually the Ebralldse, K.K., S.A. Grachev, and A.D. Mirzabekov. 1988. higher-order structure of the solenoidal helix of nucleo- A highly basic domain bound to the sharply somes, crystals of a nucleosome containing a 179-bp bent region of nucleosomal DNA. Nature 331: 365. Finch, J.T., L.C. Lutter, D. Rhodes, R.S. Brown, B. Rush- DNA fragment derived from a 5S RNA gene sequence, ton, and A. Klug. 1979. X-ray and electron microscope the four core histones, and the histone H1 globular studies on the nucleosome structure. FEBS Proc. Meet. domain expressed in bacteria have been prepared. A 51: 193. two-step in vitro reconstitution procedure has been Finch, J.T., L.C. Lutter, D. Rhodes, R.S. Brown, B. Rush- ton, M. Levitt, and A. Klug. 1977. Structure of nucleo- used to prepare these particles: (1) 179-bp core par- some core particles of chromatin. Nature 269: 29. ticles are reconstituted from c-octamers and the defined Grandy, D.K. and J.B. Dodgson. 1987. Structure and organi- sequence DNA fragment using the high to low salt zation of the chicken H2B histone gene family. Nucleic dialysis method (Thomas and Butler 1977). (2) After Acids Res. 15: 1063. purification by DEAE-HPLC, the particles sponta- Grunstein, M. 1990 Histone function in transcription. Annu. Rev. Cell Biol. 6: 643. neously form nucleosomes upon addition of the recom- Klug, A., D. Rhodes, J. Smith, J.T. Finch, and J.O. Thomas. binant globular domain of X. laevis histone H1 (Hlg) 1980. A low resolution structure for the histone core of the in low salt. The integrity of the particles was examined nucleosome. Nature 287: 509. by nondenaturing gel electrophoresis and by limited Kornberg, R.D. 1977. Structure of chromatin. Annu. Rev. Biochem. 46: 931. micrococcal nuclease digestion. This nucleosome has Kornberg, R.D. and Y. Lorch. 1992. Chromatin structure and higher mobility under electrophoresis than the corre- transcription. Annu. Rev. Cell Biol. 8: 563. sponding core particle, a feature that allows us to study Laine, B., D. Kmiecik, P. Sautiere, and G. Biserte. 1978. Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

272 RICHMOND, RECHSTEINER, AND LUGER

Primary structure of chicken erythrocyte . Crystals of a nucleosome core particle containing defined Biochimie 60: 147. sequence DNA. J. Mol. Biol. 199: 161. McGhee, J.D. and G. Felsenfeld. 1980. Nucleosome struc- Richmond, T.J., J.T. Finch, B. Rushton, D. Rhodes, and A. ture. Annu. Rev. Btochem. 49: 1115. Klug. 1984. Structure of the nucleosome core particle at 7 Mirzabekov, A.D., V.V. Shick, A.V. Belyavsky, and S.G. ,~ resolution. Nature 311: 532. Bavykin. 1978. Primary organization of nucleosome core Shick, V.V., A.V. Belyavsky, S.G. Bavykin, and A.D. Mir- particle of chromatin: Sequence of histone arrangement zabekov. 1980. Primary organization of the nucleosome along DNA. Proc. Natl. Acad. Sci. 75: 4184. core particles. Sequential arrangement of histones along Old, R.W., H.R. Woodland, J.E.M. Ballantine, T.C. AI- DNA. J. Mol Btol. 139: 491. dridge, C.A. Newton, W,A. Bains, and P.C. Turner. 1982. Simpson, R.T. and D.W. Stafford. 1983. Structural features of Organisation and expression of cloned histone gene clus- a phased nucleosome core particle. Proc. Natl. Acad. Sct. ters from Xenopus laevis and Xenopus borealis. Nucleic 80: 51. Acids Res. 23: 7561. Struck, M.-M., A. Klug, and T.J. Richmond. 1992. Com- Pederson, D.S., F. Thoma, and R.T. Simpson. 1986. Core parison of X-ray structures of the nucleosome core particle particle, fiber, and transcriptionally active chromatin struc- in two different hydration states. J. Mol. Biol. 224: 253. ture. Annu. Rev. Cell Biol. 2: 117. Studier, F.W., A.H. Rosenberg, and J.J. Dunn. 1990. Use of Pennmgs, S., G. Meersseman, and E.M. Bradbury. 1991. T7 RNA polymerase to direct the expression of cloned Mobility of positioned nucleosomes on 5S rDNA. J. Mol. genes. Methods Enzymol. 185: 60. Biol. 220: 101. Thomas, J.O. and P.J.G. Butler. 1977. Characterization of the Pruss, D. and A.P. Wolffe. 1993. Histone-DNA contacts in a octamer of histones free in solution. J. Mol. Biol. 116: 769. nucleosome core containing a Xenopus 5S rRNA gene. van Holde, K.E. 1988. Chromatin: Springer Series in Molecu- Biochemistry 32: 6810. lar Biology (ed. A. Rich). Springer-Verlag, New York. Ramakrishnan, V., J.T. Finch, V. Graziano, P.L. Lee, and Wang, S.W., A.J. Robins, R. d'Andrea, and J.R.E. Wells. R.M, Sweet. 1993. Crystal structure of globular domain of 1985. Inverted duplication of histone genes in chicken and histone H5 and its implications for nucleosome binding. disposition of regulatory sequences. Nucleic Acids Res. Nature 362: 219. 13: 1369. Rhodes, D., R.S. Brown, and A. Klug. 1989. Crystallization Wolffe, A.P. 1991. Implications of DNA replication for eu- of nucleosome core particles. Methods Enzymol. 170: 420. karyotic . J. Cell Sci. 99: 201. Richmond, T.J., M.A. Searles, and R.T. Simpson. 1988. Downloaded from symposium.cshlp.org on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press

Studies of Nucleosome Structure

T.J. Richmond, T. Rechsteiner and K. Luger

Cold Spring Harb Symp Quant Biol 1993 58: 265-272 Access the most recent version at doi:10.1101/SQB.1993.058.01.031

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