Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.chromatin structure ͉ electron microscopy ͉ linker histone ͉ reconstitution E ukaryotic chromosomes have a compact structure in which linear nucleosome arrays are first folded into a fiber of around 30-nm diameter (1, 2). The fundamental repeating unit of chromatin, the nucleosome core particle, organizes 147 bp of DNA in 1.7 left-handed superhelical turns around an octamer of the four core histones (H2A, H2B, H3, and H4) (3-5). Linker histone (H1͞H5) binding organizes an additional 20 bp of DNA to complete the nucleosome containing 167 bp of DNA (6, 7). Such binding determines the geometry of the DNA entering and exiting the nucleosome core particle (8). In nucleosome arrays, adjacent nucleosomes are separated by linker DNA, varying in length between 0 and 80 bp in a tissue-and species-specific manner (9, 10). In vitro, linear nucleosome arrays fold into the ''30-nm'' fiber upon increasing ionic strength (11) in a process that depends on both the integrity of the core histone N-terminal tails (12, 13) and the presence of the linker histone (14,15).During the past three decades evidence from EM (14-23), x-ray and neutron scattering (24-27), electric and photochemical dichroism (28-31), sedimentation analysis (32-35), nuclease digestion (6, 9, 36), and x-ray crystallography (4, 5, 37, 38) has led to the proposal of a number of different models for the 30-nm fiber. These models fall into two main classes: the one-start helix or solenoid models, and the two-start helix models. The solenoid models are comprised of simple one-start helices in which successive nucleosomes are adjacent in the filament and connected by linker DNA that bends into t...

The mechanisms by which chromatin structure decompacts to permit access to DNA are largely unknown. Here, using a model nucleosome array system reconstituted from recombinant histone octamers we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of thè 30nm' chromatin fibre. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains stoichiometric concentrations of bound linker histone, which is essential for the formation of the `30nm' chromatin fibre. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.

SUMMARY A complete, 52-protein, 2.5 million Dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between “full-length” and “deleted” forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1+ and C2+ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4+ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.