Andrew Travers

DNA bending and its relation to nucleosome positioning

X-ray and solution studies have shown that the conformation of a DNA double helix depends strongly on its base sequence. Here we show that certain sequence-dependent modulations in structure appear to determine the rotational positioning of DNA about the nucleosome. Three different experiments are described. First, a piece of DNA of defined sequence (169 base-pairs long) is closed into a circle, and its structure examined by digestion with DNAase I: the helix adopts a highly preferred configuration, with short runs of (A, T) facing in and runs of (G, C) facing out. Secondly, the same sequence is reconstituted with a histone octamer: the angular orientation around the histone core remains conserved, apart from a small uniform increase in helix twist. Finally, it is shown that the average sequence content of DNA molecules isolated from chicken nucleosome cores is non-random, as in a reconstituted nucleosome: short runs of (A, T) are preferentially positioned with minor grooves facing in, while runs of (G, C) tend to have their minor grooves facing out. The periodicity of this modulation in sequence content (10.17 base-pairs) corresponds to the helix twist in a local frame of reference (a result that bears on the change in linking number upon nucleosome formation). The determinants of translational positioning have not been identified, but one possibility is that long runs of homopolymer (dA) X (dT) or (dG) X (dC) will be excluded from the central region of the supercoil on account of their resistance to curvature.

HMG1 and 2, and related ‘architectural’ DNA-binding proteins

The HMG-box proteins, one of the three classes of high mobility group (HMG) chromosomal proteins, bend DNA and bind preferentially to distorted DNA structures. The proteins appear to act primarily as architectural facilitators in the assembly of nucleoprotein complexes; for example, in effecting recombination and in the initiation of transcription. HMG-box proteins might be targeted to particular DNA sites in chromatin by either protein-protein interactions or recognition of specific DNA structures.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p

The bromodomain is an ∼110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 Å resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15–29 of histone H4 acetylated at the ζ‐N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N‐acetyl lysine residue. Only residues 16–19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N‐acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water‐mediated H‐bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

DNA structural variations in the E. coli tyrT promoter

X-ray studies have established that the structure of a right-handed, Watson-Crick double helix can change from place to place along its length as a function of base sequence. The base pairs transmit deformations out to the phosphate backbone, where they can then be recognized by proteins and other DNA-binding reagents. Here we have examined at single-bond resolution the interactions of three commonly used nucleases (DNAase I, DNAase II, and copper-phenanthroline) with a DNA of natural origin, the 160 bp tyrT promoter. All three of these reagents seem sensitive to DNA backbone geometry rather than base sequence per se. Their sequence-dependent patterns of cleavage provide evidence for structural polymorphism of several sorts: global variation in helix groove width, global variation in radial asymmetry, and local variation in phosphate accessibility. These findings explain how sequence zones of a certain base composition, or purine-pyrimidine asymmetry, can influence the recognition of DNA by protein molecules.

DNA supercoiling - a global transcriptional regulator for enterobacterial growth?

A fundamental principle of exponential bacterial growth is that no more ribosomes are produced than are necessary to support the balance between nutrient availability and protein synthesis. Although this conclusion was first expressed more than 40 years ago, a full understanding of the molecular mechanisms involved remains elusive and the issue is still controversial. There is currently agreement that, although many different systems are undoubtedly involved in fine-tuning this balance, an important control, and in our opinion perhaps the main control, is regulation of the rate of transcription initiation of the stable (ribosomal and transfer) RNA transcriptons. In this review, we argue that regulation of DNA supercoiling provides a coherent explanation for the main modes of transcriptional control — stringent control, growth-rate control and growth-phase control — during the normal growth of Escherichia coli.

H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing.

H-NS is a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H-NS selectively silences specific genes of the bacterial chromosome. However, many studies have concluded that H-NS binds sequence-independently to DNA, leaving the molecular basis for its selectivity unexplained. We show that the negative regulatory element (NRE) of the supercoiling-sensitive Escherichia coliproU gene contains two identical high-affinity binding sites for H-NS. Cooperative binding of H-NS is abrogated by changes in DNA superhelical density and temperature. We further demonstrate that the high-affinity sites nucleate cooperative binding and establish a nucleoprotein structure required for silencing. Mutations in these sites result in loss of repression by H-NS. In this model, silencing at proU, and by inference at other genes directly regulated by H-NS, is tightly controlled by the cooperativity between bound H-NS molecules.

Position and orientation of the globular domain of linker histone H5 on the nucleosome

It is essential to identify the exact location of the linker histone within nucleosomes, the fundamental packing units of chromatin, in order to understand how condensed, transcriptionally inactive chromatin forms. Here, using a site-specific protein–DNA photo-crosslinking method, we map the binding site and the orientation of the globular domain of linker histone H5 on mixed-sequence chicken nucleosomes. We show, in contrast to an earlier model, that the globular domain forms a bridge between one terminus of chromatosomal DNA and the DNA in the vicinity of the dyad axis of symmetry of the core particle. Helix III of the globular domain binds in the major groove of the first helical turn of the chromatosomal DNA, whereas the secondary DNA-binding site on the opposite face of the globular domain of histone H5 makes contact with the nucleosomal DNA close to its midpoint. We also infer that helix I and helix II of the globular domain of histone H5 probably face, respectively, the solvent and the nucleosome. This location places the basic carboxy-terminal region of the globular domain in a position from which it could simultaneously bind the nucleosome-linking DNA strands that exit and enter the nucleosome.

High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

Priming the nucleosome: a role for HMGB proteins?

The high‐mobility‐group B (HMGB) chromosomal proteins are characterized by the HMG box, a DNA‐binding domain that both introduces a tight bend into DNA and binds preferentially to a variety of distorted DNA structures. The HMGB proteins seem to act primarily as architectural facilitators in the manipulation of nucleoprotein complexes; for example, in the assembly of complexes involved in recombination and transcription. Recent genetic and biochemical evidence suggests that these proteins can facilitate nucleosome remodelling. One mechanism by which HMGB proteins could prime the nucleosome for migration is to loosen the wrapped DNA and so enhance accessibility to chromatin‐remodelling complexes and possibly also to transcription factors. By constraining a tight loop of untwisted DNA at the edge of a nucleosome, an HMGB protein could induce movements in the contacts between certain core histones that would result in an overall change in nucleosome structure.

DNA chaperones: A solution to a persistence problem?

Andrew A. Travers, Sarbjit S. Ner, and Mair E. A. Churchill’ Medical Research Council Laboratory of Molecular Biology Cambridge CB2 2QH England *Department of Ceil and Structural Biology University of Illinois at Urbana-Champaign Urbana, Illinois 61601 In both the eukaryotic nucleus and the eubacterial nucleoid, nucleoprotein complexes establish and maintain the ordered packaging of the DNA genome. The paradigm for this mode of organization is the nucleosome core parti- cle in which the histone octamer wraps 145 bp of DNA in 1.6 left-handed superhelical turns, equivalent to an aver- age deflection of the double-helical axis by 47” for each turn of the duplex. This tight folding is in striking contrast with the solution properties of DNA, in which under physio- logical ionic conditions the DNA double helix possesses only limited flexibility. The experimentally determined per- sistence length (the length over which tangents to adjacent lengths tend to point in the same direction) of the polymer is 450-500 A or - 140 bp (reviewed by Hagerman, 1988). Such a fragment of DNA is not rigid, but can assume only a limited range of configurations and does not by itself attain a curvature comparable with that in the nucleosome core particle. Therefore, by trapping nearly two superheli- cal turns of DNA the nucleosome substantially reduces the end-to-end distance of such a DNA fragment (Figure 1). The required deformation of the double helix and the loss of entropy associated with the reduced conforma- tional freedom would be expected to incur a substantial energetic penalty on the formation of nucleosomes. The biological problem is how to build such condensed nucleoprotein structures in a chromosome. In vitro the as- sembly of the nucleosome core particle can be initiated by the histone H3/H4 tetramer that organizes 120 bp of DNA as a superhelix (Hansen et al., 1991; Hayes et al., 1991). How does this basic protein core trap the DNA in a superhelical configuration at a rate that will ensure rapid assembly? It is likely that after the initial contact with the tetramer, the DNA progressively wraps around the parti- cle. One way to facilitate this process is to prime the DNA for assembly into such a higher order complex, i.e., to prebend, pretwist, or both and to stabilize the DNA in a particular configuration that is able to transfer readily to the other proteins. Maintaining the DNA in such a configu- ration is analogous to the function of certain protein chap- erones, which stabilize a polypeptide in a conformation that is appropriate for subsequent assembly, but would be unstable without a chaperone. One such chaperone, nucleoplasmin, maintains histones in an accessible form for nucleosome assembly (Laskey et al., 1978). However, since the DNA undergoes a greater conformational change than the histones, it is likely that the optimal as-