Discrete Structural Domains Research Articles

Identification and structural analysis of a carbohydrate-binding module specific to alginate, a representative of a new family, CBM96

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35Å resolution. TM6-N4 exhibited an overall β-sandwich fold architecture with two antiparallel β-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.

CTCF maintains regulatory homeostasis of cancer pathways

BackgroundCTCF binding to DNA helps partition the mammalian genome into discrete structural and regulatory domains. Complete removal of CTCF from mammalian cells causes catastrophic genome dysregulation, likely due to widespread collapse of 3D chromatin looping and alterations to inter- and intra-TAD interactions within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction of CTCF expression are viable, albeit with increased cancer incidence. Here, we exploit chronic Ctcf hemizygosity to reveal its homeostatic roles in maintaining genome function and integrity.ResultsWe find that Ctcf hemizygous cells show modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these are enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, we observe dysregulation of the expression of several hundred genes, which are concentrated in cancer-related pathways, and are caused by changes in transcriptional regulation. Chromatin structure is preserved but some loop interactions are destabilized; these are often found around differentially expressed genes and their enhancers. Importantly, the transcriptional alterations identified in vitro are recapitulated in mouse tumors and also in human cancers.ConclusionsThis multi-dimensional genomic and epigenomic profiling of a Ctcf hemizygous mouse model system shows that chronic depletion of CTCF dysregulates steady-state gene expression by subtly altering transcriptional regulation, changes which can also be observed in primary tumors.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Cohort intelligence algorithm for discrete and mixed variable engineering problems

In this study, the performance of an emerging socio inspired metaheuristic optimization technique referred to as Cohort Intelligence (CI) algorithm is evaluated on discrete and mixed variable nonlinear constrained optimization problems. The investigated problems are mainly adopted from discrete structural optimization and mixed variable mechanical engineering design domains. For handling the discrete solution variables, a round off integer sampling approach is proposed. Furthermore, in order to deal with the nonlinear constraints, a penalty function method is incorporated. The obtained results are promising and computationally more efficient when compared to the other existing optimization techniques including a Multi Random Start Local Search algorithm. The associated advantages and disadvantages of CI algorithm are also discussed evaluating the effect of its two parameters namely the number of candidates, and sampling space reduction factor.

The functional requirement of two structural domains within telomerase RNA emerged early in eukaryotes.

Telomerase emerged during evolution as a prominent solution to the eukaryotic linear chromosome end-replication problem. Telomerase minimally comprises the catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TR) that provides the template for telomeric DNA synthesis. While the TERT protein is well-conserved across taxa, TR is highly divergent amongst distinct groups of species. Herein, we have identified the essential functional domains of TR from the basal eukaryotic species Trypanosoma brucei, revealing the ancestry of TR comprising two distinct structural core domains that can assemble in trans with TERT and reconstitute active telomerase enzyme in vitro. The upstream essential domain of T. brucei TR, termed the template core, constitutes three short helices in addition to the 11-nt template. Interestingly, the trypanosome template core domain lacks the ubiquitous pseudoknot found in all known TRs, suggesting later evolution of this critical structural element. The template-distal domain is a short stem-loop, termed equivalent CR4/5 (eCR4/5). While functionally similar to vertebrate and fungal CR4/5, trypanosome eCR4/5 is structurally distinctive, lacking the essential P6.1 stem-loop. Our functional study of trypanosome TR core domains suggests that the functional requirement of two discrete structural domains is a common feature of TRs and emerged early in telomerase evolution.

Purification and Structural Analysis of Plectin and BPAG1e.

Plectin and BPAG1e belong to the plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions. Owing to the mosaic organization of plakins, the structure of their constituent individual domains or small multi-domain segments can be analyzed isolated. Yet, understanding the integrated function of large regions, oligomers, and heterocomplexes of plakins is difficult due to the large and segmented structure. Here, we describe methods for the production of plectin and BPAG1e samples suitable for structural and biophysical analysis. In addition, we discuss the combination of hybrid methods that yield information at several resolution levels to study the complex, multi-domain, and flexible structure of plakins.

Order statistics inference for describing topological coupling and mechanical symmetry breaking in multidomain proteins

Cooperativity is a hallmark of proteins, many of which show a modular architecture comprising discrete structural domains. Detecting and describing dynamic couplings between structural regions is difficult in view of the many-body nature of protein-protein interactions. By utilizing the GPU-based computational acceleration, we carried out simulations of the protein forced unfolding for the dimer WW - WW of the all-β-sheet WW domains used as a model multidomain protein. We found that while the physically non-interacting identical protein domains (WW) show nearly symmetric mechanical properties at low tension, reflected, e.g., in the similarity of their distributions of unfolding times, these properties become distinctly different when tension is increased. Moreover, the uncorrelated unfolding transitions at a low pulling force become increasingly more correlated (dependent) at higher forces. Hence, the applied force not only breaks "the mechanical symmetry" but also couples the physically non-interacting protein domains forming a multi-domain protein. We call this effect "the topological coupling." We developed a new theory, inspired by order statistics, to characterize protein-protein interactions in multi-domain proteins. The method utilizes the squared-Gaussian model, but it can also be used in conjunction with other parametric models for the distribution of unfolding times. The formalism can be taken to the single-molecule experimental lab to probe mechanical cooperativity and domain communication in multi-domain proteins.

Late Pleistocene structural evolution of the Camarillo fold belt: Implications for lateral fault growth and seismic hazard in Southern California

The Camarillo fold belt in the Western Transverse Ranges poses a significant seismic hazard to nearly one million people living in Southern California, yet few published geologic or geochronological data from this fold belt exist. The Camarillo fold belt is composed of several south-verging, west-plunging anticlines that characterize the western extent of the Simi fault zone, which extends for 40 km through urbanized Ventura and Los Angeles Counties. Surface and subsurface geologic data are utilized to accurately construct five cross sections within discrete structural domains to assess the local style of deformation, and to quantify the magnitudes of fault slip, fault- and fold-related uplift, and percent shortening. Eight new optically stimulated luminescence (OSL) dates from three paleoseismic trenches and six numerical dates from a previous study were utilized to quantify the timing and rates of deformation on discrete faults and folds. The onset of deformation in the Camarillo fold belt is everywhere younger than ca. 125 ka and locally as young as ca. 25 ka. Quaternary deformation occurs on reactivated steeply dipping (70°) Miocene faults, with shortening being largely accommodated in a narrow zone (<2 km) of folding where fold-related uplift is typically twice as great as hanging-wall uplift in response to fault slip. A minimum fault slip rate between 0.8 mm/yr and 1.4 mm/yr, a recurrence interval of 715–1100 yr, and a maximum Mw 6.8 earthquake for faults within the Simi fault zone is estimated, which are comparable to other better-studied fold belts in Southern California. A model of punctuated lateral fault propagation is proposed to explain westward growth of the Simi fault, which occurs in discrete pulses that are separated by intervals of fault displacement accumulation and fold amplification during constant-fault-length conditions. Lateral fault growth is limited in space and time by an orthogonal north-striking fault set, which juxtaposes a series of west-plunging anticlines that decrease in structural relief and age toward the west.

Oligomerisation of the Human Cardiac Ryanodine Receptor Amino-Terminus

The cardiac ryanodine receptor (RyR2) mediates the release of calcium from the sarcoplasmic reticulum of cardiac myocytes. The functional channel is composed of four identical subunits with the C-terminal part comprising the transmembrane domain predicted to form the Ca2+-conducting pore. The large N-terminal cytoplasmic portion of RyR2 is believed to serve as a scaffold for interaction with accessory proteins, ions and other regulatory molecules. RyR2 channel gating is regulated by a complex network of inter- and intra-subunit interactions between discrete structural domains. It has been proposed that disruption of inter-domain cross-talk results in abnormal RyR2 channel function, as observed in catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure.Here, we report that the RyR2 amino-terminus, containing one of the three CPVT-associated mutation hot spots, is capable of self-association. Chemical cross-linking of an RyR2 N-terminal fragment (BT4L; residues 1-906) indicated that it can assemble into tetramers. Moreover, BT4L expressed in mammalian HEK293 cells was found to form tetramers through endogenous disulphide bonds. We undertook a site-directed mutagenesis approach to identify the cysteines involved in BT4L disulphide bond formation, whereby cysteine residues were substituted by serine. The BT4L Cys361 Ser mutant did not form DTT-sensitive tetramers, suggesting that Cys361 participates in disulphide bond formation. When BT4L was co-expressed with full-length RyR2 in HEK293 cells it translocated from the cytosol to the microsomal fraction. The functional significance of RyR2 N-terminus self-association was studied by [3H]ryanodine binding assays. We found that the BT4L fragment activates the channel at low Ca2+ concentrations most likely by disrupting inter-subunit N-terminal self-association within the tetrameric channel. Our findings suggest that the RyR2 N-terminus regulates channel function through inter-subunit interactions. This work was supported by the British Heart Foundation.

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca(2+) release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [(3)H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.

The folding cooperativity of a protein is controlled by its chain topology

The three-dimensional structures of proteins often show a modular architecture comprised of discrete structural regions or domains. Cooperative communication between these regions is important for catalysis, regulation and efficient folding; lack of coupling has been implicated in the formation of fibrils and other misfolding pathologies. How different structural regions of a protein communicate and contribute to a protein's overall energetics and folding, however, is still poorly understood. Here we use a single-molecule optical tweezers approach to induce the selective unfolding of particular regions of T4 lysozyme and monitor the effect on other regions not directly acted on by force. We investigate how the topological organization of a protein (the order of structural elements along the sequence) affects the coupling and folding cooperativity between its domains. To probe the status of the regions not directly subjected to force, we determine the free energy changes during mechanical unfolding using Crooks' fluctuation theorem. We pull on topological variants (circular permutants) and find that the topological organization of the polypeptide chain critically determines the folding cooperativity between domains and thus what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to kinetic trapping and misfolding.

Engineered toxins: New therapeutics

Clostridial neurotoxins possess discrete structural domains with distinct pharmacological properties. Aspects of neurotoxin function with therapeutic potential include specific neuronal binding, intracellular (cytosolic) delivery of biologically active protein and inhibition of SNARE-mediated secretion. Understanding the structure function relationship of the neurotoxin protein enables the creation of recombinant proteins incorporating select domains of the neurotoxins to produce novel proteins with therapeutic potential in a range of clinical applications.

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into 'neighborhoods' comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.

Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQ-binding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membrane-binding surface contains an alpha-helix and a beta-hairpin, forming a hydrophobic plateau. The UQ-flavin distance (8.5 A) is shorter than the UQ-cluster distance (18.8 A), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD.

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

NAD +-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD +-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.

Intranuclear Rodlets in the Substantia Nigra: Interactions with Marinesco Bodies, Ubiquitin, and Promyelocytic Leukemia Protein

There is growing appreciation that the nucleus is organized into an array of discrete structural domains, each subserving a specific function. These functional nuclear bodies are to be distinguished from pathological intranuclear inclusions which have been described in a variety of neurodegenerative diseases. Marinesco bodies (MBs) are eosinophilic ubiquitinated intranuclear inclusions found in pigmented neurons of the human substantia nigra and locus coeruleus. Traditionally considered non-pathological entities, more recent studies have indicated that MBs are associated with the age-associated degenerative changes in the substantia nigra and striatal loss of dopaminergic terminals. In the present morphological study of the human substantia nigra, we demonstrate colocalization, contiguity, and focally shared immunoreactivity between MBs and neuronal intranuclear rodlets (INRs). The latter nuclear structures of uncertain function are markedly decreased in the cortex of Alzheimer's disease, but not dementia with Lewy bodies. In addition, we demonstrate an interaction between INRs and promyelocytic leukemia (PML) protein, the signature protein of PML nuclear bodies. These results suggest that structures which subserve the functional compartmentalization of the neuronal nucleus may be relevant to elucidating cellular mechanisms of age-related motor dysfunction.

Active intraplate strike-slip faulting and transpressional uplift in the Mongolian Altai

Abstract The Mongolian Altai is a Late Cenozoic intraplate strike-slip deformation belt which formed as a distant strain response to the Indo-Eurasian collision over 2000 km to the south. We report results from 5 weeks of detailed fieldwork carried out during summer 2000 in northwestern Mongolia investigating the crustal architecture of the Altai at latitude 48°N. The region can be divided into discrete Cenozoic structural domains each dominated by a major dextral strike-slip fault system or range-bounding thrust fault. Gentle bends along the major strike-slip faults are marked by transpressional uplifts including asymmetric thrust ridges, restraining bends, and triangular thrust-bounded massifs. These transpressional uplifts (Tsambagarav Massif, Altun Huhey Uul, Sair Uul, Hoh Serhiyn Nuruu, Omno Hayrhan Uula, Mengildyk Nuruu) comprise the highest mountains in the Mongolian Altai and are structural and metamorphic culminations exposing polydeformed greenschist-amphibolite grade basement recording at least two phases of Palaeozoic ductile deformation overprinted by Cenozoic brittle structures. Cenozoic thrust faults with the greatest amounts of displacement bound the W and SW sides of ranges throughout the region and consistently verge WSW. Each major range is essentially a NE-tilted block and this is reflected by asymmetric internal drainage patterns. Many faults are considered active because they deform surficial deposits, form prominent scarps, and define range fronts with low sinuosity where active alluvial fan deposition takes place. Reactivation of the prevailing NW-striking, NE-dipping Palaeozoic basement anisotropy is a regionally important control on the orientation and kinematics of Cenozoic faults. At first order, the Altai is spatially partitioned into a low-angle thrust belt that overthrusts the Junggar Basin on the Chinese side and a high-angle SW-vergent dextral transpressional belt on the Mongolian side. The mechanically rigid Hangay craton and Junggar basement block which bound the Altai on either side have played a major role in focusing Late Cenozoic deformation along their boundaries and within the Altai. The geometric relationship between rigid block boundaries, Palaeozoic basement structural anisotropy, and the dominantly NE SHmax (derived from India’s continued NE indentation) has dictated the kinematics of Late Cenozoic deformation in the Altai, Gobi Altai, and Sayan regions.

Structural dynamics of the receptor-binding domain of colicin E9.

Colicin E9 is a 61 kDa antibacterial protein secreted by E. coli. In order for it to enter the cytoplasm of susceptible bacteria and kill them by hydrolysing their DNA, the colicin must first interact with an outer membrane receptor on the target cell, BtuB, and a translocation pathway involving Tol proteins. The receptor binding, translocation and DNase functions of colicin E9 are housed in discrete structural domains, which have been independently expressed and characterized. The minimal receptor-binding domain is a 76 amino acid protein (min-R). X-ray structure determination of a related colicin shows its receptor-binding-domain to have a helical hairpin structure (S. Soelaiman, K. Jakes, N. Wu, C. Li and M. Shoham, Molecular Cell. 2001, 8, 1053). Our solution NMR studies of min-R have confirmed it has a helical hairpin structure, and shown it has multiple slowly interchanging conformers and a flexible inter-helix loop. A plausible interpretation of these data is that in solution the helical hairpin can adopt a variety of structures differing in the spatial relationship of the two helices. A possible biological role for this involves the hairpin opening during translocation into bacteria.

Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries.

Eukaryotic genomes are organized into discrete structural and functional chromatin domains. Here, we show that distinct site-specific histone H3 methylation patterns define euchromatic and heterochromatic chromosomal domains within a 47-kilobase region of the mating-type locus in fission yeast. H3 methylated at lysine 9 (H3 Lys9), and its interacting Swi6 protein, are strictly localized to a 20-kilobase silent heterochromatic interval. In contrast, H3 methylated at lysine 4 (H3 Lys4) is specific to the surrounding euchromatic regions. Two inverted repeats flanking the silent interval serve as boundary elements to mark the borders between heterochromatin and euchromatin. Deletions of these boundary elements lead to spreading of H3 Lys9 methylation and Swi6 into neighboring sequences. Furthermore, the H3 Lys9 methylation and corresponding heterochromatin-associated complexes prevent H3 Lys4 methylation in the silent domain.