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1.
Wu C  Travers A 《Biochemistry》2005,44(43):14329-14334
Using a novel competition assay to determine the relative strength of different histone octamer-binding sites, we have compared three natural and two synthetic sites. We show that the relative affinities of these sites for the histone octamer depend upon both the temperature and octamer concentration. In particular, under certain conditions, a natural octamer-binding site from a yeast promoter outcompetes a synthetic sequence of comparable affinity to the strongest previously described positioning sequence. Under other conditions, this synthetic sequence is the preferred octamer ligand. We infer that sequence selection by the histone octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters may contribute differently to nucleosome formation. These findings indicate that previous studies designed to identify strong octamer-binding sites may fail to select some natural strong binding sites.  相似文献   

2.
A complex between the single-stranded DNA of the bacteriophage M13 and the histone octamer was analyzed by electron microscopy, low-angle X-ray diffraction and nuclease analysis. The morphology and the diffraction pattern of the complex strongly resemble those of the nucleosome. These results, as well as the finding of a protected DNA fragment about 100 nucleotides long following single-stranded DNA specific nuclease digestion, indicate that 'a nucleosome-like' complex can be formed between single-stranded DNA and the histone octamer. Competition experiments suggest that under physiological conditions the histone octamer is transferred from single- to double-stranded DNA.  相似文献   

3.
It has been found that histone octamer of calf thymus (H2A--H2B--H3--H4)2 can exist in two structural states--"loose" (2M NaCl) and "compact" one (4M NaCl). The compact state of the octamer is characterized by screening of part of tyrosyls for quenching effect of ions I-, longer relaxation time of tyrosyls, greater stability of histone H3 towards trypsinolysis, complete absence of interactions between histone H3 SH-groups and parachlormercuribenzoate.  相似文献   

4.
Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.  相似文献   

5.
Characterization of the octamer of histones free in solution   总被引:15,自引:0,他引:15  
The nucleosome “core protein” isolated from chromatin in high-salt solutions (2 m-NaCl) has been characterized in detail. The preparation described yields material which is stable for prolonged periods at either 4 °C or 37 °C. It has an apparent partial specific volume of 0.767 ml/g and a sedimentation coefficient (S20,w0) of 4.77 (±0.04) S. The molecular weight, determined by sedimentation equilibrium, is 107,500 (±7700), which is compatible with an octameric structure of the form (H2A)2(H2B)2(H3)2(H4)2, as previously proposed.The sedimentation coefficient and molecular weight are very similar to those of cross-linked core protein, which is known to be octameric.  相似文献   

6.
Human extracellular superoxide dismutase (EC-SOD) is involved in the defence against oxidative stress induced by the superoxide radical. The protein is a homotetramer stabilised by hydrophobic interactions within the N-terminal region. During the purification of EC-SOD from human aorta, we noticed that material with high affinity for heparin-Sepharose formed not only a tetramer but also an octamer. Analysis of the thermodynamic stability of the octamer suggested that the C-terminal region is involved in formation of the quaternary structure. In addition, we show that the octamer is composed of both aEC-SOD and iEC-SOD folding variants. The presence of the EC-SOD octamer with high affinity may represent a way to influence the local concentration of EC-SOD to protect tissues specifically sensitive to oxidative damage.  相似文献   

7.
Histone octamer transfer by a chromatin-remodeling complex   总被引:22,自引:0,他引:22  
Lorch Y  Zhang M  Kornberg RD 《Cell》1999,96(3):389-392
RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.  相似文献   

8.
Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells. The enzyme can also locate on the cell surface and bind to plasminogen, via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells. The functions of the eukaryotic enzymes on the cell surface expression (including T cells, B cells, neutrophils, monocytoes, neuronal cells and epithelial cells) are not known. Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome (STSS) never seen before in human sufferers. We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection. In this study, a 2.4-angstrom structure of the SS2 enolase is solved, revealing an octameric arrangement in the crystal. We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay. These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well. This is, to our knowledge, the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically, and the second octamer enolase structure in addition to that of Streptococcus pneumoniae. We also investigated the plasminogen binding property of the SS2 enzyme.  相似文献   

9.
Spectropolarimetric analysis of the core histone octamer and its subunits   总被引:3,自引:0,他引:3  
The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.  相似文献   

10.
The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers.  相似文献   

11.
Nucleosomal core particles containing the right- and left-handed conformations of DNA were examined for their ability to support the BZ or ZB transition. Nucleosomes were assembled onto the B- and Z-conformations of poly[d(Gm5C)] and the B-conformation of poly[d(GC)] as previously described (1). Absorbance and circular dichroic spectroscopy indicated that the DNA on all three core particle populations could undergo the conformational BZ transition. Further, the right- to left-handed transition for both poly[d(Gm5C)] and poly[d(GC)] appeared to be facilitated by the DNAs association with the histone octamer. The DNA remained associated with the protein core subsequent to the transition, and electron microscopy and sedimentation velocity analysis indicated that there were no gross changes in nucleosomal structure. However, a change in the sedimentation value of the poly[d(Gm5C)] core particles was detected when the conformation of the DNA was altered from B to Z, resulting in a lower S20,w value for the Z-form particles than for the corresponding B-form particles.  相似文献   

12.
The histone octamer, a conformationally flexible structure   总被引:1,自引:0,他引:1  
K Park  G D Fasman 《Biochemistry》1987,26(25):8042-8045
The conformation of the histone octamer complex in solution has been shown, by circular dichroism studies, to be highly dependent on the nature of the salt milieu and its concentration. In 2 M NaCl, the complex has 43.5% alpha-helix, 16% beta-sheet, and 40.5% random structure. In 2.3 M (NH4)2SO4, the octamer has 49.0% alpha-helix and 51% random structure. These results partially explain the discrepant results obtained by the X-ray analysis of crystals obtained under varying conditions.  相似文献   

13.
Nuclei of different cell lines contain protein factors interacting with octamer ATTTGCAT. Fragment k53 of kappa-gene promoter region was used as DNA-probe. The factors from lymphoid cells yield a DNA/protein complex with mobility B0. The proteins are referred to as HF-B0. The nonspecific ubiquitous factor present in many non-lymphoid cells (for instance, HeLa cells) interacts with the probe to produce a complex whose mobility is much lower. The protein NF-B0 was isolated from the nuclear extract of myeloma MOPC21 cells. It was purified by chromatography on ion exchangers, hydroxylapatite, heparin-Sepharose and affinity sorbent containing a synthetic octamer sequence. At all the steps of purification, protein fractions were chosen for their ability to interact selectively with the octamer yielding a complex with the mobility B0. As a result, NF-B0 protein (60 +/- 2)kDa was purified 6.10(4) times to the electrophoretically homogeneous state. Purified factor NF-B0 selectively interacts with the octamer.  相似文献   

14.
Gottesfeld JM  Luger K 《Biochemistry》2001,40(37):10927-10933
Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and histone octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.  相似文献   

15.
BacteriainthegeneraofRhizobium,Bradyrhizobium,AzorhizobiumandSinorhizobiumelicitrootorstemnodulesontheirspecifichostplantsandfixatmosphericnitrogentherein.Nodulationgenes(nod,nolandnoe)playimportantrolesinthesuccessfulestablishmentofthesymbiosis,yetmost…  相似文献   

16.
We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.  相似文献   

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20.
A Hamiche  R Sandaltzopoulos  D A Gdula  C Wu 《Cell》1999,97(7):833-842
Drosophila NURF is an ATP-dependent chromatin remodeling complex that contains ISWI, a member of the SWI2/SNF2 family of ATPases. We demonstrate that NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. In the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations within minutes. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. We suggest that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer.  相似文献   

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