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.