Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA.

Automated summary

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by the formation of small intranucleosomal DNA loops (i-loops), which are more efficiently formed in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs). The presence of histone tails and NT-SSBs in linker DNA strongly facilitate the formation of i-loops during transcription of the promoter-proximal region of nucleosomal DNA, leading to the arrest of RNAP. DNA footprinting analysis supports the formation of an i-loop after stalling RNAP, indicating the involvement of histone tails and NT-SSBs in i-loop formation and arrest of RNAP during transcription.