High structural stability and catalytic activity are the two principal properties of the processive elongation complex of RNA polymerase II (Pol II). The pathway leading to the formation of a stable elongation complex and the mechanisms causing dissociation of Pol II within the genes and at transcription terminators are not well understood. Differentiation between the role of elongation factors in elongation complex stability and activity and that of the Pol II itself required a simple, minimal in vitro system. In this project, we develop a novel technique that bypasses the need for protein factors to initiate transcription and obtain the elongation complex. This technique involves the direct assembly of intermediates in the elongation pathway using purified core Pol II and synthetic RNA and DNA oligonucleotides. This method allows to assess the impact of nucleic acids components by introducing changes to the oligonucleotides through their sequence, length, and pairing affinity. We have shown that the 8 nucleotides RNA:DNA hybrid is necessary and sufficient for the formation of a stable eukaryotic EC. In addition, we have observed the previously unknown ability of the RNA:DNA hybrid to negatively regulate Pol II processivity. This dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the elongation complex, and Pol II processivity.
| Lewis, Dale E A; Komissarova, Natalia; Le, Phuoc et al. (2008) DNA sequences in gal operon override transcription elongation blocks. J Mol Biol 382:843-58 |
| 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 |
| 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 |
