Mechanics of Transcription Elongation Through The Nucleosome Using Single-Molecule and Biochemical Methods

Abstract

Transcription initiation and elongation in the eukaryotic nucleus occur in the context of chromatin. DNA wrapped around the histone core in the nucleosome constitutes a major obstacle to transcription. For transcription to occur, nucleosomes must be removed from the downstream DNA template. Numerous biochemical data indicate that this nucleosome disruption is a temporary event, with nucleosomes quickly reforming in the already transcribed portion of the template. We use a combination of single-molecule approaches (Magnetic Tweezers) and biochemical methods to investigate the mechanics of transcription elongation through nucleosomes. Specifically, we have performed real-time transcription experiments with chromatin templates (208-18 DNA based) and T7 RNA Polymerase. The disassembly of 18 nucleosomes is expected to increase tether length by ∼700 nm (each nucleosome is replaced by naked DNA of ∼146 bp). We observe changes in tether length, the size of which corresponds to diassembly of single nucleosomes. We can say so far that transcription through the nucleosomal array results in disassembly of the nucleosomes. Some additional lengthening occurs as a result of changes in the geometry of the fiber (mutual disposition of nucleosomes, DNA entry-exit angles, etc.)