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Published January 18, 2022 | Published + Supplemental Material
Journal Article Open

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Abstract

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

Additional Information

© 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). Edited by Seth Darst, Rockefeller University; received July 29, 2021; accepted December 7, 2021 by Editorial Board Member Graham C. Walker. This work was supported by grants from the NIH (R01 GM102362 to D.W. and R01 GM27681 to P.B.D.). We appreciate the beamline staff for their kind support during our remote data collection. This research used resources of the Advanced Light Source (beamlines 5.0.1, 8.2.1, and 8.2.2), which is supported by the US Department of Energy (DOE), Office of Science User Facility under Contract DE-AC02-05CH11231. We also acknowledge Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, which is supported by the DOE, Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. Data Availability: All atomic coordinates and structure factors are deposited at the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB). All PDB codes are listed in SI Appendix, Table S1. Structure coordinates data have been deposited in the PDB (7RIL, 7RIQ, 7RIM, 7RIP, 7RIW, 7RIX, and 7RIY). All other study data are included in the article and/or SI Appendix. Author contributions: J.O., P.B.D., and D.W. designed research; J.O. and J.X. performed research; T.J. and J.X. contributed new reagents/analytic tools; J.O., P.B.D., and D.W. analyzed data; and J.O., J.C., P.B.D., and D.W. wrote the paper. The authors declare no competing interest. This article is a PNAS Direct Submission. S.D. is a guest editor invited by the Editorial Board. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2114065119/-/DCSupplemental.

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Additional details

Created:
September 15, 2023
Modified:
October 23, 2023