Nature of the Nucleosomal Barrier to RNA Polymerase II

Abstract

In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic inactivation (arrest) of the pol II core enzyme. To determine potential mechanisms making nucleosomes transparent to transcription in vivo, we analyzed the nature of the nucleosome-induced arrest. We found that the arrests have been detected mostly at positions of strong intrinsic pause sites of DNA. The transient pausing makes pol II vulnerable to arrest, which involves backtracking of the elongation complex for a considerable distance on DNA. The histone-DNA contacts reestablished in front of pol II stabilize backtracked conformation of the polymerase. In agreement with this mechanism, blocking of backtracking prevents nucleosome-induced arrest. Transcript cleavage factor TFIIS reactivates the backtracked complexes and promotes pol II transcription through the nucleosome. Our findings establish the crucial role of elongation factors that suppress pol II pausing and backtracking for transcription in the context of chromatin.

Arginine-phosphate salt bridges between histones and DNA: Intermolecular actuators that control nucleosome architecture

“…Monodentate contacts, in particular, are found to be strongly localized at ± 5 helical turns with respect to the central nucleosomal dyad. The placement of these contacts is commensurate with regions of the nucleosome that previous researchers [45][46][47][48]52 have associated with a high affinity for 'invasion' by DNA binding proteins, an effect that is important for active nucleosome remodeling. Specifically, the strongest resistance to DNA unzipping, or strand separation, found in single-molecule experiments 49 , occurs around the dyad and in the end regions of the nucleosome at DNA sites roughly four to five helical turns from the dyad, precisely the locations of the termini of nucleosomal DNA structures observed to be anchored by monodentate interactions in Figure 13.…”

RNA polymerase fidelity and transcriptional proofreading

“…After misincorporation, the mismatched nucleotide at position +1 of the RNA is frayed away from the template, thereby pausing RNAP. Pausing is the first step in backtracking [63,[76][77][78][79][80][81][82][83]. The frayed nucleotide then inhibits RNA extension, because it prevents NTP binding, but favors backtracking, because the bp in position +1 is disrupted.…”

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…Transcriptional pausing can not only reduce rates of mRNA production, but also recruit regulatory factors to the TEC that modify subsequent transcription (109)(110)(111)(112), function as a precursor to transcriptional arrest and termination (113,114), help to synchronize transcription and translation in prokaryotes (115), or lead to messenger splicing or polyadenylation in eukaryotes (116,117). The longlived, "stabilized pauses" that are known to play a regulatory role are often associated with the formation of RNA hairpins in the transcript (which are thought to allosterically inactivate RNAP), or with the formation of energetically weak RNA:DNA hybrids (which are thought to induce backtracking) (118).…”