Pub Date : 2024-05-15DOI: 10.1016/j.biochi.2024.05.014
Ivan Petushkov, Andrey Feklistov, Andrey Kulbachinskiy
During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.
在转录启动过程中，细菌 RNA 聚合酶（RNAP）的全酶利用专用的 σ 因子特异性地识别启动子。在转录延伸过程中，RNAP 的核心酶主要通过将 DNA 模板和 RNA 转录本稳定地锁定在主裂隙内与核酸进行非特异性相互作用。在这里，我们展示了一种合成的DNA适配体，它能被来自嗜极细菌（Deinococcus-Thermus lineage）的核心和全酶RNAP特异性识别。这种适配体能以亚纳摩尔级的亲和力与 RNAP 结合，即使在高离子强度条件下也能形成极其稳定的复合物，阻断 RNAP 与 DNA 模板的相互作用，并在转录延伸过程中抑制 RNAP 的活性。我们认为，该配合物与 RNAP 下游 DNA 结合裂隙中的一个保守位点结合，并使其处于非活性构象。这种适配体可用于结构研究以揭示 RNAP 的构象状态、RNAP 与相关因子的亲和结合以及转录抑制剂的筛选。
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Pub Date : 2023-01-24DOI: 10.1016/j.biochi.2023.01.009
Zoltán Szeltner , Györgyi Ferenc , Tünde Juhász , Zoltán Kupihár , Zoltán Váradi , Dávid Szüts , Lajos Kovács
Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.
{"title":"Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions","authors":"Zoltán Szeltner , Györgyi Ferenc , Tünde Juhász , Zoltán Kupihár , Zoltán Váradi , Dávid Szüts , Lajos Kovács","doi":"10.1016/j.biochi.2023.01.009","DOIUrl":"https://doi.org/10.1016/j.biochi.2023.01.009","url":null,"abstract":"<div><p><span><span>Guanine<span> quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the </span></span>nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model </span>oligonucleotides<span><span><span><span>. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the </span>cytosine </span>deamination product 5-hydroxyuracil and its nucleoside counterpart in </span>RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.</span></p></div>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":"214 ","pages":"Pages 33-44"},"PeriodicalIF":0.0,"publicationDate":"2023-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49670757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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