The DNA within the eukaryotic nucleus is packaged as chromatin - a tight nucleoprotein complex composed of repeating units called nucleosomes. Pol II has developed a special mechanism to transcribe through such a tightly packed DNA. This project is aimed to investigate transcription by yeast Pol II elongation complexes through uniquely positioned nucleosome core in vitro. We apply recently developed experimental system employing assembly of authentic Pol II elongation complexes and ligation of these complexes to the mononucleosome in vitro. It has been found that this system faithfully recapitulates chief properties of the promoter-dependent experimental system such as the height and general properties of the nucleosomal barrier to transcription. We demonstrate that Pol II uses unique mechanism for transcription through chromatin. Our results in this project are summarized as follows: (i) At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation by Pol II, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer, but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. (ii) Nucleosomal barrier to Pol III is very low and transcription through nucleosome in vitro occurs with only a minor pausing. Passage of Pol III through mononucleosome results in complete dissociation of histones from DNA. Thus, molecular mechanisms for chromatin transcription by Pol II and Pol III are clearly different.
| 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 |
