title: Preparation of Chromatin Templates to Study RNA Polymerase I Transcription In Vitro.
abstract: Cellular DNA is packaged into chromatin, which is the substrate of all DNA-dependent processes in eukaryotes. The regulation of chromatin requires specialized enzyme activities to allow the access of sequence-specific binding proteins and RNA polymerases. In order to dissect chromatin-dependent features of transcription regulation in detail, in vitro systems to generate defined chromatin templates for transcription are required. I present a protocol that allows the assembly of nucleosomes on ribosomal RNA (rRNA) minigenes by salt gradient dialysis and subsequent sucrose gradient centrifugation. This procedure yields high nucleosome occupancy and high dynamic response in subsequent transcriptional analysis. It provides an invaluable tool to study rRNA gene transcription, as transcription on free DNA is clearly different from the more in vivo-like transcription on reconstituted chromatin templates.
authors: Biochemistry III, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany. gernot.laengst@ur.de.
authors: Gernot
authors: G
authors: Längst
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D003956:Dialysis; D004330:Drosophila; D005786:Gene Expression Regulation; D020459:Genes, rRNA; D006657:Histones / Q000378:metabolism; D066298:In Vitro Techniques; D009707:Nucleosomes / Q000378:metabolism; D012318:RNA Polymerase I / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D012318:RNA Polymerase I
keywords: Chromatin assembly; In vitro reconstitution; In vitro transcription; Ribosomal DNA; Salt gradient dialysis