Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET.

Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET).

Experimental analysis of the mechanism of chromatin remodeling by RNA polymerase II.

The vital process of transcription by RNA polymerase II (Pol II) occurs in chromatin environment in eukaryotic cells; in fact, moderately transcribed genes retain nucleosomal structure. Recent studies suggest that chromatin structure presents a strong barrier for transcribing Pol II in vitro, and that DNA-histone interactions are only partially and transiently disrupted during transcript elongation on moderately active genes. Furthermore, elongating Pol II complex is one of the major targets during gene regulation.

Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3 and H4. The mechanism of nucleosome survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival.