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.

Potentiometric Determination of Acid Dissociation Constants (pKa) for an Anticancer Pyrrole-Imidazole Polyamide

“…Inspired by naturally occurring netropsin and distamycin A, , Dervan and co-workers pioneered the design and development of pyrrole–imidazole (Py-Im) polyamides as a class of cell-permeable small molecules that bind to the minor groove of double-stranded DNA (dsDNA) in a sequence-specific fashion. − A set of pairing rules describes the recognition of Watson–Crick base pairs by pairs of these heterocyclic amino acids within the minor groove via the formation of distinct hydrogen bonds: Im/Py pairs distinguish G/C from C/G base pairs, whereas Py/Py pairs are degenerate for both A/T and T/A base pairs (also called W base pairs). , Py-Im polyamides can induce an expansion of the minor groove and a corresponding compression in the opposing major groove, , thereby interfering the DNA–protein interaction , and the transcriptional machinery. , The application of these synthetic molecules in biological systems leads to altered expression of cancer-related genes, including PSA, TGFβ1, NF-κB, MMP-9, ABCA1, EVI1, LOX-1, and Plk1 . In particular, Dervan’s group reported the eight-ring hairpin-shaped Py-Im polyamide PIP-1 targeting the androgen response element (ARE) half-site 5′-WGWWCW-3′, which can disrupt the interaction of ARE with androgen receptor (AR) or glucocorticoid receptor (GR) and shows efficacy against enzalutamide-resistant VCaP and LREX′ prostate cancer models in cell culture and xenografts. , Furthermore, Lown and co-workers achieved great progress in the design and synthesis of unit modules mimicking pyrrole and imidazole moieties in biological applications. − …”

“…6,7 Py-Im polyamides can induce an expansion of the minor groove and a corresponding compression in the opposing major groove, 8,9 thereby interfering the DNA−protein interaction 10,11 and the transcriptional machinery. 12,13 The application of these synthetic molecules in biological systems leads to altered expression of cancer-related genes, including PSA, 10 TGFβ1, 14 NF-κB, 15 MMP-9, 16 ABCA1, 17 EVI1, 18 LOX-1, 19 and Plk1. 20 In particular, Dervan's group reported the eight-ring hairpin-shaped Py-Im polyamide PIP-1 targeting the androgen response element (ARE) half-site 5′-WGWWCW-3′, which can disrupt the interaction of ARE with androgen receptor (AR) or glucocorticoid receptor (GR) and shows efficacy against enzalutamide-resistant VCaP and LREX′ prostate cancer models in cell culture and xenografts.…”