Assay of the Fate of the Nucleosome During Transcription by RNA Polymerase II

Abstract

This chapter discusses assaying of the fate of the nucleosome during transcription by RNA polymerase II. A novel approach for analysis of Pol II elongation complexes (ECs) employs an assembly of “authentic” ECs using histidine-tagged yeast Pol II and synthetic RNA and DNA oligonucleotides. The structure and functional properties of the assembled and promoter-initiated ECs are very similar. In this system, the fate of nucleosomes during transcription can be analyzed after ligation of the ECs to positioned mononucleosomes that are assembled separately. Nucleosomes form an absolute block to Pol II however this barrier can be reduced by increasing the ionic strength of the reaction, thus allowing more templates to be transcribed. Transcription through nucleosomes by Pol II induces the loss of an H2A/H2B dimer without changing the position of the histones on the DNA. Use of this experimental system for analysis of the fate of the nucleosome during transcription is described below. This “minimal” model system can be easily adopted for analysis of polynucleosomal templates and for analysis of the role of different elongation factors during transcription through chromatin.