RESUMO
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.
Assuntos
DNA/química , Conformação de Ácido Nucleico , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Imidazóis/química , Modelos Moleculares , Pirróis/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes.
Assuntos
DNA/genética , Ácidos Nucleicos Peptídicos/genética , Transcrição Gênica/genética , Apoptose/genética , Sítios de Ligação/genética , Replicação do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Expressão Gênica/genética , Humanos , Neoplasias/genética , Conformação de Ácido Nucleico , Proteínas Virais/genéticaRESUMO
R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template DNA strand. R-loops occur naturally in all kingdoms of life, and they have multiple biological effects. Therefore, it is of interest to study the artificial induction of R-loops and to monitor their effects in model in vitro systems to learn mechanisms. Here we describe transcription blockage in vitro by R-loop formation induced by peptide nucleic acid (PNA) binding to the non-template DNA strand.