A topoisomerase from chicken erythrocyte nuclei which does not assemble nucleosome core particles in vitro

1982 ◽  
Vol 60 (6) ◽  
pp. 651-658 ◽  
Author(s):  
Michael J. Ellison ◽  
David E. Pulleyblank
Keyword(s):  
Ionic Strength ◽  
Homogeneous Distribution ◽  
Chicken Erythrocyte ◽  
Nucleosome Assembly ◽  
Core Particle ◽  
Dna Complexes ◽  
Particle Assembly ◽  
Nucleosome Core ◽  
Nuclease Digestion

We have examined the ability of a topoisomerase purified from chicken erythrocyte nuclei to mediate nucleosome core assembly in vitro at physiological ionic strength (0.15 M NaCl). Although we have detected limited amounts of spontaneously assembled nucleosome cores at this salt concentration, the addition of this topoisomerase does not increase the amount of assembly observed. Nucleosome assembly was assayed by quantitating the amount of core particle length DNA accumulated with time upon the nuclease digestion of histone–DNA complexes. In addition, the amount of negative supercoils introduced into relaxed closed circular DNA upon nucleosome core particle assembly was determined. Correctly assembled complexes do not protect more DNA from nuclease digestion than random histone–DNA complexes but shift the heterogeneous size distribution of protected fragments to a more homogeneous distribution centred around 145 base pairs. Under our conditions of nucleosome assembly, a second histone–DNA complex which is distinct from the nucleosome core can be detected under physiological ionic strength conditions. This particle does not form in high salt assembly experiments. Similarly, the assembly of this particle is unaffected by the presence or absence of topoisomerase.

scholarly journals In vitro core particle and nucleosome assembly at physiological ionic strength.

1979 ◽  
Vol 76 (7) ◽  
pp. 3284-3288 ◽  
Author(s):  
A. Ruiz-Carrillo ◽  
J. L. Jorcano ◽  
G. Eder ◽  
R. Lurz
Keyword(s):  
Ionic Strength ◽  
Nucleosome Assembly ◽  
Core Particle

scholarly journals X-ray structure of the MMTV-A nucleosome core

2016 ◽  
Vol 113 (5) ◽  
pp. 1214-1219 ◽  
Author(s):  
Timothy D. Frouws ◽  
Sylwia C. Duda ◽  
Timothy J. Richmond
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Promoter Sequence ◽  
Core Particle ◽  
Nucleosome Core Particle ◽  
Particle Structure ◽  
X Ray ◽  
Nucleosome Core ◽  
Sequence Dependent

The conformation of DNA bound in nucleosomes depends on the DNA sequence. Questions such as how nucleosomes are positioned and how they potentially bind sequence-dependent nuclear factors require near-atomic resolution structures of the nucleosome core containing different DNA sequences; despite this, only the DNA for two similar α-satellite sequences and a sequence (601) selected in vitro have been visualized bound in the nucleosome core. Here we report the 2.6-Å resolution X-ray structure of a nucleosome core particle containing the DNA sequence of nucleosome A of the 3′-LTR of the mouse mammary tumor virus (147 bp MMTV-A). To our knowledge, this is the first nucleosome core particle structure containing a promoter sequence and crystallized from Mg2+ ions. It reveals sequence-dependent DNA conformations not seen previously, including kinking into the DNA major groove.

scholarly journals The prenucleosome, a stable conformational isomer of the nucleosome

2015 ◽  
Vol 29 (24) ◽  
pp. 2563-2575 ◽  
Author(s):  
Jia Fei ◽  
Sharon E. Torigoe ◽  
Christopher R. Brown ◽  
Mai T. Khuong ◽  
George A. Kassavetis ◽  
...  
Keyword(s):  
Motor Protein ◽  
Upstream Region ◽  
Entry And Exit ◽  
Core Particle ◽  
Nucleosome Core Particle ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Conformational Isomer

Chromatin comprises nucleosomes as well as nonnucleosomal histone–DNA particles. Prenucleosomes are rapidly formed histone–DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone–DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone–DNA species in the cell.

scholarly journals Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

1988 ◽  
Vol 8 (7) ◽  
pp. 2884-2895 ◽  
Author(s):  
G Conway ◽  
J Wooley ◽  
T Bibring ◽  
W M LeStourgeon
Keyword(s):  
Rna Processing ◽  
Rna Splicing ◽  
Protein Composition ◽  
Physical Parameters ◽  
Core Particle ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Nuclear Ribonucleoprotein ◽  
Nascent Transcripts

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.

Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles

1988 ◽  
Vol 8 (7) ◽  
pp. 2884-2895
Author(s):  
G Conway ◽  
J Wooley ◽  
T Bibring ◽  
W M LeStourgeon
Keyword(s):  
Rna Processing ◽  
Rna Splicing ◽  
Protein Composition ◽  
Physical Parameters ◽  
Core Particle ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Nuclear Ribonucleoprotein ◽  
Nascent Transcripts

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.

Influence of the media ionic strength on the formation and in vitro biological performance of polycation-DNA complexes

2021 ◽  
pp. 117930
Author(s):  
Filipe Coelho ◽  
Claudia Botelho ◽  
Juan L. Paris ◽  
Eduardo F. Marques ◽  
Bruno F. B. Silva
Keyword(s):  
Ionic Strength ◽  
Dna Complexes ◽  
The Media ◽  
Biological Performance
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