The nature of the nucleosomal barrier to transcription: Direct observation of paused intermediates by electron cryomicroscopy

Jan Bednar; Vasily M. Studitsky; Sergei A. Grigoryev; Gary Felsenfeld; Christopher L. Woodcock

doi:10.1016/S1097-2765(00)80339-1

The nature of the nucleosomal barrier to transcription: Direct observation of paused intermediates by electron cryomicroscopy

Jan Bednar, Vasily M. Studitsky, Sergei A. Grigoryev, Gary Felsenfeld, Christopher L. Woodcock

Research output: Contribution to journal › Article › peer-review

76 Scopus citations

Abstract

Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to ~20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

Original language	English
Pages (from-to)	377-386
Number of pages	10
Journal	Molecular Cell
Volume	4
Issue number	3
DOIs	https://doi.org/10.1016/S1097-2765(00)80339-1
State	Published - Sep 1999

Keywords

Binding Sites
Chromatin/metabolism
Cryoelectron Microscopy
DNA Footprinting
DNA-Directed RNA Polymerases/metabolism
DNA/metabolism
Histones/metabolism
Image Processing, Computer-Assisted
Models, Genetic
Models, Structural
Molecular Conformation
Nucleosomes/metabolism
Protein Binding
Transcription, Genetic

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10.1016/S1097-2765(00)80339-1

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title = "The nature of the nucleosomal barrier to transcription: Direct observation of paused intermediates by electron cryomicroscopy",

abstract = "Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to ~20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.",

keywords = "Binding Sites, Chromatin/metabolism, Cryoelectron Microscopy, DNA Footprinting, DNA-Directed RNA Polymerases/metabolism, DNA/metabolism, Histones/metabolism, Image Processing, Computer-Assisted, Models, Genetic, Models, Structural, Molecular Conformation, Nucleosomes/metabolism, Protein Binding, Transcription, Genetic",

author = "Jan Bednar and Studitsky, {Vasily M.} and Grigoryev, {Sergei A.} and Gary Felsenfeld and Woodcock, {Christopher L.}",

year = "1999",

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doi = "10.1016/S1097-2765(00)80339-1",

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T1 - The nature of the nucleosomal barrier to transcription

T2 - Direct observation of paused intermediates by electron cryomicroscopy

AU - Bednar, Jan

AU - Studitsky, Vasily M.

AU - Grigoryev, Sergei A.

AU - Felsenfeld, Gary

AU - Woodcock, Christopher L.

PY - 1999/9

Y1 - 1999/9

N2 - Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to ~20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

AB - Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to ~20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

KW - Binding Sites

KW - Chromatin/metabolism

KW - Cryoelectron Microscopy

KW - DNA Footprinting

KW - DNA-Directed RNA Polymerases/metabolism

KW - DNA/metabolism

KW - Histones/metabolism

KW - Image Processing, Computer-Assisted

KW - Models, Genetic

KW - Models, Structural

KW - Molecular Conformation

KW - Nucleosomes/metabolism

KW - Protein Binding

KW - Transcription, Genetic

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U2 - 10.1016/S1097-2765(00)80339-1

DO - 10.1016/S1097-2765(00)80339-1

M3 - Article

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AN - SCOPUS:0033197561

SN - 1097-2765

VL - 4

SP - 377

EP - 386

JO - Molecular Cell

JF - Molecular Cell

IS - 3

ER -

The nature of the nucleosomal barrier to transcription: Direct observation of paused intermediates by electron cryomicroscopy

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Keywords

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