Mechanism of Nucleosome Dissociation Produced by Transcription Elongation in a Short Chromatin Template

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Abstract
We have used a linear DNA template (239 bp) containing a nucleosome positioning sequence (NX1) downstream of the T7 RNA polymerase promoter to study the mechanism of transcription elongation through a nucleosome. Under ionic strength approaching physiological conditions we have observed that transcription causes nucleosome dissociation and histone redistribution within the template. We have examined the role of the different elements that, in principle, could induce nucleosome dissociation during transcription. The high affinity of histones for single-stranded DNA observed in titration experiments performed using the purified (+) and (-) strands of the NX1 fragment suggests that nucleosome dissociation is not due to the formation of segments of single-stranded DNA by RNA polymerase in the elongation process. Furthermore, our results show that although RNA can interact with core histones, the synthesized RNA is not bound to the histones dissociated by transcription. Our results indicate that core histones released during transcription can be bound to naked DNA and chromatin (with or without histones H1-H5). From the dynamic properties of excess histones bound to chromatin, we suggest a nucleosome transcription mechanism in which displaced histones are transiently bound to chromatin and finally are reassembled with DNA after the passage of the polymerase.