Construction, analysis, and transcription of model nucleosomal templates.
Transcription through the nucleosome by Saccharomyces cerevisiae RNA polymerase II (Pol II) is characterized by an almost absolute block to transcription at physiological ionic strength and displacement of one H2A/H2B dimer to form a hexasome [Mol. Cell 9 (2002) 541]. In previous studies of Pol II transcription through chromatin, templates containing nucleosomes in multiple positions were used. These templates do not allow detailed analysis of the mechanism of transcription through chromatin. Here, we describe the development of a new template that is only long enough to accommodate a single nucleosome position along the DNA so that all of the templates are identical and allow for more in-depth analysis. After ligation of the nucleosome to promoter DNA or assembled elongation complexes, the mechanism of transcription through this uniquely positioned nucleosome by various RNA polymerases can be analyzed.
Walter
Wendy
W
Department of Biochemistry and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Studitsky
Vasily M
VM
D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
D009707:Nucleosomes; D004247:DNA; D012319:RNA Polymerase II; D004262:DNA Restriction Enzymes