RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif

Abstract

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy.Py-Im polyamide | transcription inhibition | minor groove | DNA I n eukaryotes, precursor mRNA synthesis is catalyzed by the RNA polymerase II holoenzyme (pol II), which frequently pauses during transcription elongation (1-3). In addition to regulatory factors that control pol II processivity, various obstacles encountered by pol II can also lead to stalling, and even backtracking, on the DNA template (4). Factors that affect pol II transcription elongation dynamics include intrinsic DNA sequences or structures (5, 6), endogenous epigenetic DNA modifications (7), embedded ribonucleotides (8), DNA lesions (9, 10), small-molecule DNA-binders (11, 12), DNA-binding proteins including nucleosomes (13), and even pol II itself (14, 15). Transient transcriptional pausing or short-lived transcriptional blockage can be rescued by the recruitment of transcription factor IIS (TFIIS) (16, 17), a transcription factor that facilitates the cleavage of backtracked transcript. In contrast, prolonged transcriptional arrest by some bulky DNA lesions triggers either ubiquitination and degradation of pol II, or transcription-coupled nucleotide excision repair, a special DNA repair pathway that preferentially repairs DNA lesions in the transcribed strand (17, 18). Structural, genetic, and biochemical studies have greatly advanced our understanding of how pol II copes with different kinds of covalent DNA lesions caused by oxidation (19, 20), alkylation (21-24), and photocyclo-addition (10). Less well understood are the interactions of the pol II machinery when confronted with a steric blockade by small molecules bound noncovalently in the minor groove of DNA (25-27).Pyrrole-imidazole (Py-Im) polyamides are a class of small molecules that c...

A Pyrrole-Imidazole Polyamide Is Active against Enzalutamide-Resistant Prostate Cancer

“…Long term treatment of LREX’ cells with ARE-1 also decreases nascent RNA synthesis. In biophysical experiments, polyamides can halt RNAP2 elongation directly upstream of a polyamide binding site (19). We hypothesize this stalling of RNAP2 promotes ubiquitination and degradation of RPB1, ultimately interfering with RNA synthesis, which may contribute to efficacy against treatment refractory CaP.…”

Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis

“…Our systematic investigations of alkylation at different positions of thymine base suggested a potentially unique recognition motif of RNA pol II that may serve as a gatekeeper for monitoring the structural integrity of DNA minor groove. Our previous studies unveiled a couple of important recognition motifs in RNA pol II that target the DNA major-groove modifications or bulky, helix-distorting lesions during translocation (10,11,58,59). Furthermore, we recently identified two important residues (Rpb 1 R1386/H1387) in switch 1 region that are important for the early detection of minor-groove DNA binder Py-Im molecules even before it reaches the downstream bubble edge (59).…”

The Road Not Taken with Pyrrole-Imidazole Polyamides: Off-Target Effects and Genomic Binding

“…Nonetheless, one can safely posit that epigenetic changes are intimately linked to the rise of off-target effects. This is well-corroborated with the fact that small conformational distortions are sufficient to retard RNA polymerase II [17] and potentially displace histones [44]. Evidence also suggests that lipophilicity of a PIP is associated in tumor-directed delivery in situ [22], albeit there may be some dependence on the type and origin of tumors [45].…”

“…Despite the simplicity behind its chemistry, the amide bonds in PIP backbones permit the conjugation of a variety of different reactive moieties that diversify the structure and functionalities of PIPs. Their innate ability to bind the DNA minor groove allows them to be used to repress gene transcription via triggering RNA polymerase II inhibition [17], without having to resort to unstable or complex experimental procedures such RNAi or CRISPR/Cas. As such, by binding the minor groove, one can avoid the messiness of conventional small molecular inhibitors that work on protein surfaces.…”

“…PIPs have been used in a variety of biological applications; in 2005, Beerman and colleagues successfully demonstrated the ability to cause DNA damage by conjugating 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz(e)indole (CBI), an alkylating agent, to disrupt and inhibit SV40 DNA replication [18]; conjugation of other functional groups have also led to the rise of additional functionalities for PIPs, such as artificial nucleases [19], gene switches [20], transcriptional regulators via histone acetyltransferase activators [21] and imaging probes [22] upon the conjugation of fluorescent moieties (Figure 2a) or radioisotopes [23]. Even without those modifications, PIPs are still able to retain their high specificity, unlike mainstream DNA-damaging agents such as cisplatin [17].…”