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

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