Methods in enzymology最新文献_第9页

Single-molecule observation of G-quadruplex and R-loop formation induced by transcription. 单分子观测转录诱导的 G 型四联体和 R 型环的形成。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2024.01.001

Jihee Hwang, Bradleigh Palmer, Sua Myong

引用次数: 0

PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step. PAR-dCLIP：通过加入解帽步骤，检测结合在 5'末端的 RNA 结合蛋白目标转录本。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-07 DOI: 10.1016/bs.mie.2024.08.003

Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano

{"title":"PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step.","authors":"Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano","doi":"10.1016/bs.mie.2024.08.003","DOIUrl":"10.1016/bs.mie.2024.08.003","url":null,"abstract":"RNA binding proteins (RBPs) are responsible for facilitating a wealth of post-transcriptional gene regulatory functions. The role of an RBP on regulated transcripts can be investigated through a pull-down of the RBP and high-throughput sequencing (HTS) of the associated transcripts. Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), is one such pull-down method that isolates, detects, and sequences the cDNA of RBP-associated transcripts. PAR-CLIP relies on a photoactivatable ribonucleoside analogue, 4-thiouridine, to facilitate covalent RNA-protein crosslinks at 365 nm. These crosslinks permit stringent wash conditions and result in T to C mismatch incorporations during reverse transcription, a unique parameter for the computational analysis of high-confidence binding sites. However, until now, RBPs that bind at the 5'-termini of RNAs have been uniquely restricted from the full potential bandwidth of autoradiographic detection and HTS library preparation. The 5'-termini of RNAs are highly modified, including the most common Pol-II derived modification: the 7-methylguanosine (m7G) cap. In the conventional PAR-CLIP protocol, cap-binding proteins protect the m7G cap from the RNase treatment that generates the necessary substrate for autoradiographic detection and 5' adapter ligation-thus occluding entire populations of RNA from visualization and HTS. Here, we introduce decapping-PAR-CLIP or PAR-dCLIP. We incorporate a decapping step into the PAR-CLIP protocol to generate the necessary substrate to sequence m7G capped transcripts. While PAR-dCLIP was originally targeted towards known m7G-cap binding proteins, we argue that all RBP inquiries, and particularly those suspected to regulate translation, should incorporate this decapping step to ensure that all possible populations of bound transcripts are identified.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"159-222"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery, isolation, and characterization of diazeniumdiolate siderophores. 发现、分离和鉴定重氮二硫酸盐苷元。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-07-20 DOI: 10.1016/bs.mie.2024.06.006

Melanie Susman, Jin Yan, Christina Makris, Alison Butler

引用次数: 0

The production of siderophore analogues using precursor-directed biosynthesis. 利用前体定向生物合成法生产苷酸类似物。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-07-23 DOI: 10.1016/bs.mie.2024.06.009

Tomas Richardson-Sanchez, Thomas J Telfer, Cho Z Soe, Kate P Nolan, Michael P Gotsbacher, Rachel Codd

{"title":"The production of siderophore analogues using precursor-directed biosynthesis.","authors":"Tomas Richardson-Sanchez, Thomas J Telfer, Cho Z Soe, Kate P Nolan, Michael P Gotsbacher, Rachel Codd","doi":"10.1016/bs.mie.2024.06.009","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.06.009","url":null,"abstract":"Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"702 ","pages":"121-145"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142000399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

RADD: A real-time FRET-based biochemical assay for DNA deaminase studies. RADD：基于实时 FRET 的 DNA 脱氨酶生化测定。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-27 DOI: 10.1016/bs.mie.2024.08.001

Christopher A Belica, Patricia C Hernandez, Michael A Carpenter, Yanjun Chen, William L Brown, Reuben S Harris, Hideki Aihara

引用次数: 0

Analysis of mitochondrial protein aggregation and disaggregation. 线粒体蛋白质聚集和分解分析。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-20 DOI: 10.1016/bs.mie.2024.07.048

Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland

{"title":"Analysis of mitochondrial protein aggregation and disaggregation.","authors":"Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland","doi":"10.1016/bs.mie.2024.07.048","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.048","url":null,"abstract":"Deficits of mitochondrial functions have been identified in many human pathologies, in particular in age-related human neurodegenerative diseases. Hence, the molecular causes for mitochondrial dysfunction and potential protection mechanisms have become a major topic in modern cell biology. Apart from defects in their structural integrity, problems in mitochondrial protein biogenesis, including polypeptide transport, folding and assembly to active enzymes, all may result in some degree of functional defects of the organelle. An accumulation of misfolded polypeptides inside mitochondria, confounded by the dual source of mitochondrial polypeptides, will result in the formation of protein aggregates. Such aggregate accumulation bears a cell-toxic potential, resulting in mitochondrial and correlated cellular damages, summarized in the term \"aggregate proteotoxicity\". Here, we discuss methods to analyze protein aggregation in the mitochondrial matrix compartment. We also address techniques to characterize the biochemical mechanisms that reduce aggregate proteotoxicity, the disaggregation or resolubilization of aggregated polypeptides and the sequestration and neutralization of mitochondrial aggregates at specific sites inside a cell.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"475-498"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564294","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking. 通过二硫键形成和半胱氨酸特异性交联分析线粒体蛋白质的转运。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mie.2024.07.057

Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann

{"title":"Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking.","authors":"Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann","doi":"10.1016/bs.mie.2024.07.057","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.057","url":null,"abstract":"Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"257-298"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells. 链特异性 PCR 竞争性复制和加合物旁路测定法，用于评估 DNA 加合物如何干扰哺乳动物细胞中的 DNA 复制。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-10 DOI: 10.1016/bs.mie.2024.07.013

Jun Yuan, Chen Wang, Xiaomei He, Yinsheng Wang

引用次数: 0

Monitoring mitochondrial precursor processing and presequence peptide degradation. 监测线粒体前体加工和前序肽降解。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-10 DOI: 10.1016/bs.mie.2024.07.018

Cansu Kücükköse, F-Nora Vögtle, Annette Flotho

{"title":"Monitoring mitochondrial precursor processing and presequence peptide degradation.","authors":"Cansu Kücükköse, F-Nora Vögtle, Annette Flotho","doi":"10.1016/bs.mie.2024.07.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.018","url":null,"abstract":"The maturation of mitochondrial presequence precursor proteins after their import into the organelle is a complex process that requires the interaction of several mitochondrial proteases. Precursor processing by the mitochondrial presequence proteases is directly coupled to the proteolytic turnover of the cleaved targeting signal by mitochondrial presequence peptidases. Dysfunction of these enzymes is associated with a variety of human diseases, including neurological disorders, cardiomyopathies and renal diseases. In this chapter, we describe experimental approaches to study the activity of the major mitochondrial presequence protease (MPP) and of the presequence peptidases. In vitro assays and soluble mitochondrial extracts allow the assessment and experimental manipulation of peptidase and protease activity using immunoblotting, fluorescence measurements and autoradiography as readouts. In particular, the assays allow manipulation at multiple levels including in vivo, in organello or in soluble extracts/in vitro. Purification of the yeast heterodimeric MPP allows in vitro reconstitution of the initial presequence processing step using radiolabeled precursors as substrates. Application of soluble mitochondrial extracts enables direct assessment of MPP processing and presequence peptide turnover which can be easily manipulated and is uncoupled from protein translocation across the mitochondrial membranes. The techniques presented in this chapter allow in-depth analysis of precursor processing and presequence turnover as well as direct assessment of the impact of patient mutations on the activity of the presequence processing machinery.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"193-213"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of mitochondrial translation using click chemistry. 利用点击化学分析线粒体翻译。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mie.2024.07.044

Roya Yousefi, Sven Dennerlein

引用次数: 0