scholarly journals Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

2015 ◽  
Vol 112 (5) ◽  
pp. E410-E419 ◽  
Author(s):  
Celine Walmacq ◽  
Lanfeng Wang ◽  
Jenny Chong ◽  
Kathleen Scibelli ◽  
Lucyna Lubkowska ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Dna Lesions ◽  
Abasic Site ◽  
Pol Ii ◽  
Dna Lesion ◽  
Trigger Loop ◽  
Pyrimidine Dimers ◽  
Bulky Dna Lesions

In human cells, the oxidative DNA lesion 8,5′-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5′) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5′-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

scholarly journals Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

2018 ◽  
Vol 115 (11) ◽  
pp. E2538-E2545 ◽  
Author(s):  
Wei Wang ◽  
Celine Walmacq ◽  
Jenny Chong ◽  
Mikhail Kashlev ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Lesions ◽  
Structural Motif ◽  
Structural Basis ◽  
Dependent Manner ◽  
Abasic Site ◽  
Structural Explanation ◽  
Pol Ii ◽  
Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

Structural Biology of RNA Polymerase II Transcription: 20 Years On

2020 ◽  
Vol 36 (1) ◽  
pp. 1-34 ◽  
Author(s):  
Sara Osman ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Focal Point ◽  
Dna Lesions ◽  
Cellular Regulation ◽  
Pol Ii ◽  
Associated Proteins ◽  
Rna Polymerase Ii Transcription

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

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

2016 ◽  
Vol 113 (44) ◽  
pp. 12426-12431 ◽  
Author(s):  
Liang Xu ◽  
Wei Wang ◽  
Deanna Gotte ◽  
Fei Yang ◽  
Alissa A. Hare ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Minor Groove ◽  
Dna Lesions ◽  
Pol Ii ◽  
Dna Minor Groove ◽  
Minor Groove Binders ◽  
Groove Binders

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.

scholarly journals High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

2016 ◽  
Author(s):  
Chenxi Qiu ◽  
Olivia C. Erinne ◽  
Jui Dave ◽  
Ping Cui ◽  
Huiyan Jin ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Genetic Interactions ◽  
Substrate Selection ◽  
Phenotypic Data ◽  
Trigger Loop ◽  
Allele Specific ◽  
Individual Residue

The active site of multicellular RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three major mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

scholarly journals Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

2020 ◽  
Vol 295 (28) ◽  
pp. 9583-9595
Author(s):  
Aleksei Agapov ◽  
Artem Ignatov ◽  
Matti Turtola ◽  
Georgiy Belogurov ◽  
Daria Esyunina ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Stress Responses ◽  
Active Site ◽  
Rna Synthesis ◽  
Conformational Dynamics ◽  
Dna Lesions ◽  
Trigger Loop ◽  
Nucleotide Addition

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

scholarly journals Opposite roles of transcription elongation factors Spt4/5 and Elf1 in RNA polymerase II transcription through B-form versus non-B DNA structures

2021 ◽  
Author(s):  
Jun Xu ◽  
Jenny Chong ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trinucleotide Repeat ◽  
Transcription Elongation ◽  
Dna Lesions ◽  
Cag Repeat ◽  
Elongation Factors ◽  
Pol Ii ◽  
Dna Structures

Abstract Transcription elongation can be affected by numerous types of obstacles, such as nucleosome, pausing sequences, DNA lesions and non-B-form DNA structures. Spt4/5 and Elf1 are conserved transcription elongation factors that promote RNA polymerase II (Pol II) bypass of nucleosome and pausing sequences. Importantly, genetic studies have shown that Spt4/5 plays essential roles in the transcription of expanded nucleotide repeat genes associated with inherited neurological diseases. Here, we investigate the function of Spt4/5 and Elf1 in the transcription elongation of CTG•CAG repeat using an in vitro reconstituted yeast transcription system. We found that Spt4/5 helps Pol II transcribe through the CTG•CAG tract duplex DNA, which is in good agreement with its canonical roles in stimulating transcription elongation. In sharp contrast, surprisingly, we revealed that Spt4/5 greatly inhibits Pol II transcriptional bypass of CTG and CAG slip-out structures. Furthermore, we demonstrated that transcription elongation factor Elf1 individually and cooperatively with Spt4/5 inhibits Pol II bypass of the slip-out structures. This study uncovers the important functional interplays between template DNA structures and the function of transcription elongation factors. This study also expands our understanding of the functions of Spt4/5 and Elf1 in transcriptional processing of trinucleotide repeat DNA.

scholarly journals Structural basis of human transcription–DNA repair coupling

Nature ◽  
2021 ◽  
Vol 598 (7880) ◽  
pp. 368-372
Author(s):  
Goran Kokic ◽  
Felix R. Wagner ◽  
Aleksandar Chernev ◽  
Henning Urlaub ◽  
Patrick Cramer
Keyword(s):  
Dna Repair ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
Protein A ◽  
Elongation Factor ◽  
Dna Lesions ◽  
Structural Basis ◽  
Elongation Complex ◽  
Pol Ii ◽  
Dna Lesion

AbstractTranscription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription–repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

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

2022 ◽  
Vol 119 (3) ◽  
pp. e2114065119
Author(s):  
Juntaek Oh ◽  
Tiezheng Jia ◽  
Jun Xu ◽  
Jenny Chong ◽  
Peter B. Dervan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Minor Groove ◽  
Dna Lesions ◽  
Structural Basis ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
X Ray ◽  
A Minor ◽  
Insight Into

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.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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