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.
Highlights
These features of Py-Im oligomers led to several studies on the inhibition of specific gene expression in colon, cervical, and prostate cancer [35,36,37,38,39,40]
We solved seven X-ray crystal structures, including a double-stranded DNA complexed with hairpin Py-Im 1 and four different polymerase II (Pol II) elongation complexes bound with Py-Im 1
We previously showed that hairpin Py-Im 1 (Fig. 1A) induces consecutive Pol II pausing/arrest using the scaffolds containing a fully matched transcription bubble [29]
Summary
Py-Im Induces Strong Transcriptional Pausing at Both Full-Bubble Scaffold and Miniscaffold. As the first step toward obtaining the structural insights into Py-Im–induced Pol II arrest, we tested whether we could recapitulate these Py-Im–induced pausing/arrests using the miniscaffold for structural studies To this end, we designed a miniscaffold harboring a hairpin Py-Im 1 binding sequence (50-TGACCA-30) (Fig. 1B). The binding of hairpin Py-Im 1 leads to minor groove widening as well as major groove compression of the DNA duplex in a similar manner as cyclic Py-Im molecules (SI Appendix, Fig. S1C) [33, 34, 42, 43] This high-resolution structure hairpin Py-Im–dsDNA complex was used as a starting model for building the downstream Py-Im–dsDNA region of the Pol II EC–Py-Im complex and paved the way for our structural studies of Py-Im–induced Pol II–arrested complexes. In the Py-Im–dsDNA structure, we found that the hydrogen bonding network interactions between the γ-turn moiety of hairpin Py-Im 1 (α-ammonium tip) and the DNA minor groove are mediated by two ordered water molecules
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