Abstract
We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.
Highlights
It has long been appreciated that nucleosomes form a strong blockade to transcript elongation by polymerase II (pol II) in vitro [1, 2]
Removal of the N-terminal histone tails resulted in facilitated transcription through a nucleosome by both yeast and human pol II, an effect most noticeable at lower ionic strength (40 mM KCl)
For human pol II, tail removal reduced nucleosome-specific pausing over the entire length of the 603 nucleosome, leading to both increased traversal and the accumulation of complexes paused at more distal locations
Summary
Protein Purification—Yeast pol II with a hexahistidinetagged RPB3 subunit was purified as described [2]. (Note that 603 and 603R are identical sequences; they were constructed to ligate to the transcription complex in opposite orientations.) Yeast pol II elongation complexes were assembled on a 50-bp DNA fragment from 9-nucleotide RNA and separate template and non-template strands. These complexes were immobilized on nickel-nitrilotriacetic acid-agarose beads, washed, eluted from the beads, and ligated to nucleosomal templates or the equivalent non-nucleosomal DNA. The 603 and 603R templates were assembled into nucleosomes by salt dialysis, bound to streptavidin beads, and assembled into pol II preinitiation complexes with purified pol II and TFIIH and recombinant TATA box-binding protein, TFIIB, TFIIE, and TFIIF as described [24]. For both yeast and human transcription reactions, labeled transcripts were resolved by electrophoresis on denaturing gels and quantified using a Storm Imager and ImageQuant software
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