scholarly journals Different Proteins Mediate Step-wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

2020 ◽  
Author(s):  
Hugo Maruyama ◽  
Eloise I. Prieto ◽  
Takayuki Nambu ◽  
Chiho Mashimo ◽  
Kosuke Kashiwagi ◽  
...  
Keyword(s):  
Higher Order ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromosomal Proteins ◽  
Pyrobaculum Calidifontis ◽  
Archaeal Species ◽  
Nuclease Digestion ◽  
Chromosomal Architecture ◽  
Order Structures

AbstractArchaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

scholarly journals The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Antoine Hocher ◽  
Maria Rojec ◽  
Jacob B Swadling ◽  
Alexander Esin ◽  
Tobias Warnecke
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coli Strain ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromatin Architecture ◽  
Archaeal Histone

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

Evidence for Nucleosomal Phasing and a Novel Protein Specifically Binding to Cucumber Satellite DNA

1994 ◽  
Vol 49 (1-2) ◽  
pp. 79-86 ◽  
Author(s):  
Thilo C. Fischer ◽  
Sabine Groner ◽  
Ulrike Zentgraf ◽  
Vera Hemleben
Keyword(s):  
Satellite Dna ◽  
Specific Binding ◽  
Apparent Molecular Weight ◽  
Micrococcal Nuclease ◽  
Type I ◽  
Dna Repeats ◽  
Cucumis Sativus L ◽  
Nuclease Digestion

The nucleosomal organization and the protein-binding capability of highly repeated and methylated satellite DNA of cucumber (Cucumis sativus L.), comprising approx. 30% of the genome, were analyzed. Nucleosomal core DNA from satellite type I was prepared after micrococcal nuclease digestion of chromatin and sequenced. Most of the core sequences obtained could be grouped in two main (A and B) and two minor groups (C and D) indicating a specific and complex phasing of nucleosomes on this satellite DNA. In vitro, gel retardation assays with cloned satellite DNA repeats (types I-IV) demonstrated a specific binding of nuclear proteins. These specific binding effects are also obtained with genomic, in vivo methylated and sequence heterogeneous (1 to 10% diversity) satellite type I DNA. For the first time in plants, a satellite DNA-binding protein with an apparent molecular weight of 14 kDa (SAT 14) was identified.

The role of Bni5 in the regulation of septin higher-order structure formation

2015 ◽  
Vol 396 (12) ◽  
pp. 1325-1337 ◽  
Author(s):  
Csilla Patasi ◽  
Jana Godočíková ◽  
Soňa Michlíková ◽  
Yan Nie ◽  
Radka Káčeriková ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Structural Changes ◽  
Higher Order ◽  
Cytoskeletal Proteins ◽  
Order Structure ◽  
Division Plane ◽  
Cellular Processes ◽  
Order Structures

Abstract Septins are a family of conserved cytoskeletal proteins playing an essential role in cytokinesis and in many other cellular processes in fungi and animals. In budding yeast Saccharomyces cerevisiae, septins form filaments and higher-order structures at the mother-bud neck depending on the particular stage of the cell cycle. Septin structures at the division plane serve as a scaffold to recruit the proteins required for particular cellular processes. The formation and localization of septin structures at particular stages of the cell cycle also determine functionality of these proteins. Many different proteins participate in regulating septin assembly. Despite recent developments, we are only beginning to understand how specific protein-protein interactions lead to changes in the polymerization of septin filaments or assembly of higher-order structures. Here, using fluorescence and electron microscopy, we found that Bni5 crosslinks septin filaments into networks by bridging pairs or multiple filaments, forming structures that resemble railways. Furthermore, Bni5 appears to be a substrate of the Elm1 protein kinase in vitro. Moreover, Elm1 induces in the presence of Bni5 disassembly of long septin filaments, suggesting that these proteins may participate in the hourglass to double ring transition. This work gives new insight into the regulatory role of Bni5 in the structural changes of septins.

scholarly journals Structure of active chromatin: higher-order folding of transcriptionally active chromatin in control and hypothyroid rat liver

1997 ◽  
Vol 322 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Kulbhushan TIKOO ◽  
Q. Anwar HAMID ◽  
Ziledar ALI
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Sedimentation Rate ◽  
Higher Order ◽  
Micrococcal Nuclease ◽  
Percentage Increase ◽  
Active Chromatin ◽  
Nuclease Digestion ◽  
Rate Of Sedimentation ◽  
Cd Studies

Investigations have been carried out into the salt-induced higher-order folding in the transcriptionally active chromatin region of rat liver nuclei by nuclease digestion, sedimentation and CD. The sensitivity of active chromatin in nuclei to micrococcal nuclease was suppressed by raising the ionic strength from 25 to 90 mM, indicating the occurrence of salt-induced condensation. The rate of sedimentation of fractionated inactive chromatin fragments of both moderate (∼3.5 kbp) and large (∼8.8 kbp) size increased maximally to the same extent, while that of active chromatin fragments was dependent on their size. The rate of sedimentation of moderately sized active chromatin fragments (∼5.5 kbp) showed a maximal 15% increase at 90 mM ionic strength. In contrast, a large increase (at least 60%) in the sedimentation rate of large active chromatin fragments (∼21 kbp) was observed at 65 mM ionic strength. A reasonable degree of higher-order folding was observed in large active chromatin fragments even at 25 mM ionic strength. On considering the percentage increase in sedimentation rate as a measure of the higher-order folding of chromatin, a different type of higher-order folding was observed in active chromatin fragments. Although the percentage increase in sedimentation decreased from 40 to 24% with an increase in the size of active chromatin from ∼3 to ∼9 kbp, a further increase in size up to 16 kbp brought the percentage increase back to 40%. CD studies agreed with the conclusions drawn from sedimentation studies. Active chromatin from hypothyroid rats showed similar folding behaviour, but the order of folding was slightly lower than for control active chromatin, at all sizes.

scholarly journals Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure

1984 ◽  
Vol 219 (1) ◽  
pp. 165-171 ◽  
Author(s):  
L Schiaffonati ◽  
L Bardella ◽  
G Cairo ◽  
V Giancotti ◽  
A Bernelli-Zazzera
Keyword(s):  
Acute Phase ◽  
Chromatin Structure ◽  
Rna Synthesis ◽  
Elongation Rate ◽  
Micrococcal Nuclease ◽  
Chromosomal Proteins ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Liver Nuclei ◽  
Phase Process

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.

scholarly journals Parathymosin Affects the Binding of Linker Histone H1 to Nucleosomes and Remodels Chromatin Structure

2005 ◽  
Vol 280 (16) ◽  
pp. 16143-16150 ◽  
Author(s):  
Goran Martic ◽  
Zoe Karetsou ◽  
Katerina Kefala ◽  
Anastasia S. Politou ◽  
Cedric R. Clapier ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Histone H1 ◽  
Higher Order ◽  
Linker Histone ◽  
Laser Scanning Microscopy ◽  
Micrococcal Nuclease ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Linker Histone H1

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1in vitroandin vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using anin vitrochromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.

scholarly journals LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior

2020 ◽  
Author(s):  
Nana Naetar ◽  
Konstantina Georgiou ◽  
Christian Knapp ◽  
Irena Bronshtein ◽  
Elisabeth Zier ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Higher Order ◽  
Lamin A ◽  
Independent Manner ◽  
Nuclear Interior ◽  
Order Structures

AbstractLamins form stable filaments at the nuclear periphery in metazoans. Unlike B-type lamins, lamins A and C localize also in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). We show that lamin A in the nuclear interior is formed from newly expressed pre-lamin A during processing and from soluble mitotic mature lamins in a LAP2α-independent manner. Binding of LAP2α to lamins A/C in the nuclear interior during interphase inhibits formation of higher order structures of lamin A/C in vitro and in vivo, keeping lamin A/C in a mobile low assembly state independent of lamin A/C S22 phosphorylation. Loss of LAP2α causes formation of larger, less mobile and biochemically stable lamin A/C structures in the nuclear interior, which reduce the mobility of chromatin. We propose that LAP2α is essential to maintain a mobile lamin A/C pool in the nuclear interior, which is required for proper nuclear functions.

scholarly journals Architecture of the Mto1/2 microtubule nucleation complex

2019 ◽  
Author(s):  
Harish C. Thakur ◽  
Eric M. Lynch ◽  
Weronika E. Borek ◽  
Xun X. Bao ◽  
Sanju Ashraf ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Higher Order ◽  
Microtubule Nucleation ◽  
Cooperative Interactions ◽  
Dimerization Interface ◽  
Functional Version ◽  
Complex Architecture ◽  
Order Structures

ABSTRACTProteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators ofγ-tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of theSchizosaccharomyces pombeCM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complexin vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimersin vitroand that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutantsin vivo.

scholarly journals Testing an Electrostatic Interaction Hypothesis of Hepatitis B Virus Capsid Stability by Using an In Vitro Capsid Disassembly/Reassembly System

2009 ◽  
Vol 83 (20) ◽  
pp. 10616-10626 ◽  
Author(s):  
Margaret Newman ◽  
Pong Kian Chua ◽  
Fan-Mei Tang ◽  
Pei-Yi Su ◽  
Chiaho Shih
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Electrostatic Interaction ◽  
Micrococcal Nuclease ◽  
Interaction Hypothesis ◽  
Micrococcal Nuclease Digestion ◽  
B Virus ◽  
Nuclease Digestion ◽  
Capsid Stability

ABSTRACT To test a previously coined “charge balance hypothesis” of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecular-weight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined “electrostatic interaction hypothesis” is discussed.

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