Regulation of the interaction of non -coding RNAs with RNA polymerase II

RNA Polymerase II (Pol II) transcription of mRNA is a highly regulated process that is central to gene expression. Mouse B2 and human Alu RNA are noncoding RNAs that bind Pol II and repress mRNA transcription. These RNAs possess separate Pol II binding and transcription repression domains. Mouse B1 RNA and human scAlu RNA are two other ncRNAs that also bind Pol II but do not inhibit transcription. B1 RNA and scAlu RNA possess Pol II binding domains but lack repression domains. We investigated how ncRNA binding to Pol II is controlled by other factors in order to understand the interplay between binding Pol II and the ability to repress transcription. TFIIF was found to associate with B1 RNA/Pol II and scAlu/Pol II complexes and decreases their kinetic stability. Holo-TFIIF was required to observe dissociation of ncRNA/Pol II complexes as the individual subunits of TFIIF were not sufficient. The instability of these ncRNA/Pol II/TFIIF complexes is abrogated when the ncRNAs contain transcriptional repression domains.;HeLa nuclear extracts possess an activity that dissociates B2 RNA from Pol II. We developed a chromatography scheme to purify this dissociation activity to near homogeneity, and used mass spectrometry to identify proteins in the purified fraction. Ongoing studies are aimed at determining which protein(s) has the activity. Biochemical studies were performed to understand the mechanism by which the purified factor facilitates the dissociation of B2 RNA from Pol II. The purified factor could also dissociate Alu RNA from Pol II. TFIIF was found to increase the factor's ability to carry out its dissociation activity. Dissociation activity required all four NTPs. From mechanistic experiments addressing the NTP dependence of dissociation, we arrived at a model whereby Pol II binds B2 RNA, extends its 3' end, and the purified factor then dissociates the extended B2 RNA from Pol II.