Pub Date : 2024-07-18DOI: 10.1038/s41594-024-01361-z
Ilias Skeparnias, Jinwei Zhang
NEAT1 long noncoding RNA orchestrates paraspeckle assembly and impacts tumorigenesis, fertility and immunity. Its maturation requires RNase P cleavage yielding an unstable transfer RNA-like multiple endocrine neoplasia-β tRNA-like transcript (menRNA) due to CCACCA addition. Here we report the crystal structure of human menRNA, which partially mimics tRNAs to drive RNase P and ELAC2 processing. Biophysical analyses uncover an RNA-centric, riboswitch-like mechanism whereby the nascent CCA reshapes the RNA folding landscape and propels a spontaneous conformational isomerization that directs repeat CCA addition, marking the menRNA and defective tRNAs for degradation. This study reveals the mechanisms of NEAT1 lncRNA maturation and menRNA biogenesis and uncovers an RNA-centric, riboswitch-like mechanism where menRNA drives its own conformational isomerization that directs repeat CCA addition and rapid degradation.
NEAT1 长非编码 RNA 可协调副颈组装，影响肿瘤发生、生育和免疫。它的成熟需要 RNase P 的裂解，由于 CCACCA 的添加，会产生不稳定的类似转移 RNA 的多发性内分泌肿瘤-β tRNA 样转录物（menRNA）。我们在此报告了人类 menRNA 的晶体结构，它部分模拟 tRNA 驱动 RNase P 和 ELAC2 处理。生物物理分析发现了一种以 RNA 为中心的类似核糖开关的机制，新生的 CCA 重塑了 RNA 的折叠结构，并推动了自发的构象异构化，从而引导重复的 CCA 加成，将 menRNA 和有缺陷的 tRNA 标记为降解。
{"title":"Structural basis of NEAT1 lncRNA maturation and menRNA instability","authors":"Ilias Skeparnias, Jinwei Zhang","doi":"10.1038/s41594-024-01361-z","DOIUrl":"10.1038/s41594-024-01361-z","url":null,"abstract":"NEAT1 long noncoding RNA orchestrates paraspeckle assembly and impacts tumorigenesis, fertility and immunity. Its maturation requires RNase P cleavage yielding an unstable transfer RNA-like multiple endocrine neoplasia-β tRNA-like transcript (menRNA) due to CCACCA addition. Here we report the crystal structure of human menRNA, which partially mimics tRNAs to drive RNase P and ELAC2 processing. Biophysical analyses uncover an RNA-centric, riboswitch-like mechanism whereby the nascent CCA reshapes the RNA folding landscape and propels a spontaneous conformational isomerization that directs repeat CCA addition, marking the menRNA and defective tRNAs for degradation. This study reveals the mechanisms of NEAT1 lncRNA maturation and menRNA biogenesis and uncovers an RNA-centric, riboswitch-like mechanism where menRNA drives its own conformational isomerization that directs repeat CCA addition and rapid degradation.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1650-1654"},"PeriodicalIF":12.5,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-15DOI: 10.1038/s41594-024-01358-8
Pedro Beltrao
The idea of a scientific discovery is often linked to the eureka moment of a lone scientist, which then transforms our thinking. However, scientific discoveries are never made by individuals in isolation. They build on the work of countless researchers, and often require interdisciplinary and collaborative teams of researchers.
{"title":"The power of scientific collaborations and the future of structural biology","authors":"Pedro Beltrao","doi":"10.1038/s41594-024-01358-8","DOIUrl":"10.1038/s41594-024-01358-8","url":null,"abstract":"The idea of a scientific discovery is often linked to the eureka moment of a lone scientist, which then transforms our thinking. However, scientific discoveries are never made by individuals in isolation. They build on the work of countless researchers, and often require interdisciplinary and collaborative teams of researchers.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1309-1310"},"PeriodicalIF":12.5,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141618205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-08DOI: 10.1038/s41594-024-01350-2
Rohit Roy, Hashim M. Al-Hashimi
AlphaFold 3 represents a breakthrough in predicting the 3D structures of complexes directly from their sequences, offering insights into biomolecular interactions. Extending predictions to molecular behavior and function requires a shift from viewing biomolecules as static 3D structures to dynamic conformational ensembles.
{"title":"AlphaFold3 takes a step toward decoding molecular behavior and biological computation","authors":"Rohit Roy, Hashim M. Al-Hashimi","doi":"10.1038/s41594-024-01350-2","DOIUrl":"10.1038/s41594-024-01350-2","url":null,"abstract":"AlphaFold 3 represents a breakthrough in predicting the 3D structures of complexes directly from their sequences, offering insights into biomolecular interactions. Extending predictions to molecular behavior and function requires a shift from viewing biomolecules as static 3D structures to dynamic conformational ensembles.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 7","pages":"997-1000"},"PeriodicalIF":12.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-08DOI: 10.1038/s41594-024-01347-x
Agnel Sfeir
Curiosity-driven and fundamental discovery science must be justified in its importance to human health and translational potential for practical applications and cures. However, many groundbreaking discoveries occur through the freedom to ask fundamental questions — the how and why — without knowing where they lead. Presented here is an example of a clinical target that emerged from a seemingly simple question in chromosome biology.
{"title":"Obscure DNA sequences unveil a new cancer target","authors":"Agnel Sfeir","doi":"10.1038/s41594-024-01347-x","DOIUrl":"10.1038/s41594-024-01347-x","url":null,"abstract":"Curiosity-driven and fundamental discovery science must be justified in its importance to human health and translational potential for practical applications and cures. However, many groundbreaking discoveries occur through the freedom to ask fundamental questions — the how and why — without knowing where they lead. Presented here is an example of a clinical target that emerged from a seemingly simple question in chromosome biology.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1311-1312"},"PeriodicalIF":12.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01340-4
Ilias Skeparnias, Charles Bou-Nader, Dimitrios G. Anastasakis, Lixin Fan, Yun-Xing Wang, Markus Hafner, Jinwei Zhang
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic ‘elbow’ feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation. The authors uncover a Père David’s deer-like design for long noncoding RNAs such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which partially mimics the transfer RNA (tRNA) structure to recruit select tRNA processing enzymes for maturation and to create novel regulatory RNAs such as the MALAT1-associated small cytoplasmic RNA.
{"title":"Structural basis of MALAT1 RNA maturation and mascRNA biogenesis","authors":"Ilias Skeparnias, Charles Bou-Nader, Dimitrios G. Anastasakis, Lixin Fan, Yun-Xing Wang, Markus Hafner, Jinwei Zhang","doi":"10.1038/s41594-024-01340-4","DOIUrl":"10.1038/s41594-024-01340-4","url":null,"abstract":"The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic ‘elbow’ feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation. The authors uncover a Père David’s deer-like design for long noncoding RNAs such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which partially mimics the transfer RNA (tRNA) structure to recruit select tRNA processing enzymes for maturation and to create novel regulatory RNAs such as the MALAT1-associated small cytoplasmic RNA.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1655-1668"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01345-z
Léa Mammri, Paul T. Conduit
Microtubules within cells often have 13 protofilaments but are nucleated by multi-protein y-TuRCs complexes that display 14 γ-tubulin molecules. High-resolution cryo-EM structures of γ-TuRCs after nucleation show that these γ-TuRCs ‘close’ during nucleation to display only 13 γ-tubulin molecules for protofilament assembly.
{"title":"Cryo-EM structures of γ-TuRC reveal molecular insights into microtubule nucleation","authors":"Léa Mammri, Paul T. Conduit","doi":"10.1038/s41594-024-01345-z","DOIUrl":"10.1038/s41594-024-01345-z","url":null,"abstract":"Microtubules within cells often have 13 protofilaments but are nucleated by multi-protein y-TuRCs complexes that display 14 γ-tubulin molecules. High-resolution cryo-EM structures of γ-TuRCs after nucleation show that these γ-TuRCs ‘close’ during nucleation to display only 13 γ-tubulin molecules for protofilament assembly.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 7","pages":"1004-1006"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01344-0
Marina C. Nocente, Anida Mesihovic Karamitsos, Emilie Drouineau, Manon Soleil, Waad Albawardi, Cécile Dulary, Florence Ribierre, Hélène Picaud, Olivier Alibert, Joël Acker, Marie Kervella, Jean-Christophe Aude, Nick Gilbert, Françoise Ochsenbein, Sophie Chantalat, Matthieu Gérard
The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50–80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4–subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs. Here, the authors show that the canonical BRG/BRM-associated factor (SWI/SNF) chromatin remodeler generates subnucleosomes containing 50–80 bp of DNA associated with the four core histones. These hemisome-like particles interact with OCT4 to expand its binding domain at enhancers.
典型的 BRG/BRM 相关因子（cBAF）复合物对哺乳动物细胞中增强子的染色质开放至关重要。然而，开放染色质的性质仍不清楚。在这里，我们发现除了产生无组蛋白的 DNA 外，cBAF 还能产生稳定的半球状亚核糖体颗粒，其中包含与 50-80 bp DNA 相关的四个核心组蛋白。我们的全基因组分析表明，cBAF通过靶向和分裂脆弱的核小体来产生这些颗粒。在小鼠胚胎干细胞中，这些亚核小体成为主转录因子OCT4的体内结合底物，与OCT4 DNA基序的存在无关。在增强子上，OCT4与亚核小体的相互作用增加了OCT4的占据率，与无组蛋白DNA占据的区域相比，OCT4结合的基因组间隔扩大了一个数量级。我们认为，依赖于cBAF的亚核小体协调了一种分子机制，将OCT4在染色质开放中的功能投射到其DNA基团之外。
{"title":"cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers","authors":"Marina C. Nocente, Anida Mesihovic Karamitsos, Emilie Drouineau, Manon Soleil, Waad Albawardi, Cécile Dulary, Florence Ribierre, Hélène Picaud, Olivier Alibert, Joël Acker, Marie Kervella, Jean-Christophe Aude, Nick Gilbert, Françoise Ochsenbein, Sophie Chantalat, Matthieu Gérard","doi":"10.1038/s41594-024-01344-0","DOIUrl":"10.1038/s41594-024-01344-0","url":null,"abstract":"The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50–80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4–subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs. Here, the authors show that the canonical BRG/BRM-associated factor (SWI/SNF) chromatin remodeler generates subnucleosomes containing 50–80 bp of DNA associated with the four core histones. These hemisome-like particles interact with OCT4 to expand its binding domain at enhancers.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1756-1768"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01DOI: 10.1038/s41594-024-01336-0
Wenxin Hu, Amit Kumar, Syed Faraz Ahmed, Shijiao Qi, David K. G. Ma, Honglin Chen, Gurjeet J. Singh, Joshua M. L. Casan, Michelle Haber, Ilia Voskoboinik, Matthew R. McKay, Joseph A. Trapani, Paul G. Ekert, Mohamed Fareh
The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects. This study uses single-base tiled screens, bioinformatics, comprehensive mutagenesis and proteomics to provide a high-resolution view of RNA silencing with PspCas13b. It reveals design principles for potent silencing without collateral effects.
{"title":"Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing","authors":"Wenxin Hu, Amit Kumar, Syed Faraz Ahmed, Shijiao Qi, David K. G. Ma, Honglin Chen, Gurjeet J. Singh, Joshua M. L. Casan, Michelle Haber, Ilia Voskoboinik, Matthew R. McKay, Joseph A. Trapani, Paul G. Ekert, Mohamed Fareh","doi":"10.1038/s41594-024-01336-0","DOIUrl":"10.1038/s41594-024-01336-0","url":null,"abstract":"The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects. This study uses single-base tiled screens, bioinformatics, comprehensive mutagenesis and proteomics to provide a high-resolution view of RNA silencing with PspCas13b. It reveals design principles for potent silencing without collateral effects.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1702-1716"},"PeriodicalIF":12.5,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01336-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01DOI: 10.1038/s41594-024-01349-9
Ruth M. Saecker, Andreas U. Mueller, Brandon Malone, James Chen, William C. Budell, Venkata P. Dandey, Kashyap Maruthi, Joshua H. Mendez, Nina Molina, Edward T. Eng, Laura Y. Yen, Clinton S. Potter, Bridget Carragher, Seth A. Darst
During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70–RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation. Time-resolved cryo-EM captured transient intermediates during E. coli RNAP promoter melting, revealing conformational changes affecting stepwise transcription bubble opening. Results inform how DNA sequence controls bacterial transcription initiation.
在细菌 RNA 聚合酶（RNAPs）形成转录功能开放复合物（RPo）的过程中，瞬时中间产物在克服限速步骤之前会堆积起来。目前还没有关于这些相互转化的实时结构描述。为了填补这一空白，我们在此使用时间分辨低温电子显微镜（cryo-EM）捕捉大肠杆菌σ70-RNAP与λPR启动子混合后120毫秒或500毫秒内产生的四个中间产物。低温电子显微镜快照显示，转录泡的上游边缘迅速解除配对，随后两个保守的非模板链（nt-strand）碱基逐步插入 RNAP 口袋。随着 nt 链 "读出 "的延伸，RNAP 夹闭，将抑制性 σ70 结构域从活性位点裂隙中排出。模板链在 120 毫秒前完全未配对，但仍保持动态，这表明在随后的步骤中，未知的构象变化完成了 RPo 的形成。鉴于这些事件很可能描述了许多细菌启动子的 DNA 开启过程，本研究提供了有关 DNA 序列如何调控 RPo 形成步骤的见解。
{"title":"Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy","authors":"Ruth M. Saecker, Andreas U. Mueller, Brandon Malone, James Chen, William C. Budell, Venkata P. Dandey, Kashyap Maruthi, Joshua H. Mendez, Nina Molina, Edward T. Eng, Laura Y. Yen, Clinton S. Potter, Bridget Carragher, Seth A. Darst","doi":"10.1038/s41594-024-01349-9","DOIUrl":"10.1038/s41594-024-01349-9","url":null,"abstract":"During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70–RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation. Time-resolved cryo-EM captured transient intermediates during E. coli RNAP promoter melting, revealing conformational changes affecting stepwise transcription bubble opening. Results inform how DNA sequence controls bacterial transcription initiation.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1778-1788"},"PeriodicalIF":12.5,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-25DOI: 10.1038/s41594-024-01341-3
Philipp Throll, Luciano G. Dolce, Palma Rico-Lastres, Katharina Arnold, Laura Tengo, Shibom Basu, Stefanie Kaiser, Robert Schneider, Eva Kowalinski
Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA-binding mode for m3C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors. The authors use cryo-electron microscopy and biochemistry to reveal how 3-methylcytosine (m3C) methyltransferases bind tRNA. They also find that the human m3C methyltransferase METTL6 forms a tRNA-dependent complex with seryl-tRNA synthetase to methylate target tRNAs efficiently.
{"title":"Structural basis of tRNA recognition by the m3C RNA methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase","authors":"Philipp Throll, Luciano G. Dolce, Palma Rico-Lastres, Katharina Arnold, Laura Tengo, Shibom Basu, Stefanie Kaiser, Robert Schneider, Eva Kowalinski","doi":"10.1038/s41594-024-01341-3","DOIUrl":"10.1038/s41594-024-01341-3","url":null,"abstract":"Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA-binding mode for m3C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors. The authors use cryo-electron microscopy and biochemistry to reveal how 3-methylcytosine (m3C) methyltransferases bind tRNA. They also find that the human m3C methyltransferase METTL6 forms a tRNA-dependent complex with seryl-tRNA synthetase to methylate target tRNAs efficiently.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 10","pages":"1614-1624"},"PeriodicalIF":12.5,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01341-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：