Nucleosomes are """"""""transparent"""""""" to transcription by RNA polymerase II (Pol II) in vivo, but not in vitro. The nature of the strong in vitro barrier to Pol II is unknown. Nucleosomes survive transcription in vivo, but passage of Pol II generates an """"""""open"""""""" chromatin state where a high-order nucleosome structure is disturbed. During transcription, the individual nucleosomes change their position on DNA, some histones dissociate from the histone octamer, and the histone tails become chemically modified. The relationship between all these alterations and the strength of the nucleosomal barrier is mostly circumstantial. This is due to the lack of an in vitro system where the biochemical details of the process can be analyzed. We accomplished this goal by designing the """"""""minimal"""""""" in vitro system where core enzymes of the yeast Pol II, Pol III, and E. coli RNA polymerase (RNAP) transcribe through a positioned mononucleosome.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Kireeva, Maria L; Hancock, Brynne; Cremona, Gina H et al. (2005) Nature of the nucleosomal barrier to RNA polymerase II. Mol Cell 18:97-108
Studitsky, Vasily M; Walter, Wendy; Kireeva, Maria et al. (2004) Chromatin remodeling by RNA polymerases. Trends Biochem Sci 29:127-35
Walter, W; Kireeva, M L; Tchernajenko, V et al. (2003) Assay of the fate of the nucleosome during transcription by RNA polymerase II. Methods Enzymol 371:564-77
Walter, Wendy; Kireeva, Maria L; Studitsky, Vasily M et al. (2003) Bacterial polymerase and yeast polymerase II use similar mechanisms for transcription through nucleosomes. J Biol Chem 278:36148-56
Kireeva, Maria L; Lubkowska, Lucyna; Komissarova, Natalia et al. (2003) Assays and affinity purification of biotinylated and nonbiotinylated forms of double-tagged core RNA polymerase II from Saccharomyces cerevisiae. Methods Enzymol 370:138-55