Both transcription initiation and elongation can be regulated by factors that directly bind to RNAPII.We are studying the repressive interaction of human RNAPII with ncRNAs (in collaboration with James Goodridge, UC Boulder), and by the RecQL5 helicase. RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-coding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-electron microscopy structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.
Gene transcription is regulated in response to environmental changes as well as developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain non-coding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II, but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. We have obtained cryo-EM reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, also shared with a previously characterized yeast aptamer, is close to the active center and thus in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNA analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.
Kassube SA, Fang J, Grob P, Yakovchuk P, Goodrich JA, Nogales E. (2013) Structural insights into Transcriptional Repression by non-coding RNAs that bind to Human Pol II. J Mol Biol, 425,3639-3648.
Kassube, S.A., Jinek, M., Fang, J., Tsutakawa, S. and Nogales, E. (2013) Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5. Nat. Struct. Mol. Biol., 20(7)892-9.