| In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
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Author:
Date:
2021-04-05
[Abstract] The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously ...
[摘要] [摘要]大多数细胞蛋白的s的降解通过26S在真核生物蛋白酶。但是，内在无序的蛋白质（IDPs）包含大量的非结构化区域，并且内在地不稳定，因此很容易通过不依赖泛素的20S蛋白酶体降解。越来越多的证据最近显示ň植物境内流离失所者的平衡也可以通过20S蛋白酶控制。但是，由于缺乏用于体外分离20S蛋白酶体和降解测定的系统协议，因此我们对植物中IDP和20S蛋白酶体降解的功能和调控机制的研究和理解一直处于婴儿期与其他生物。在这里，我们通过采用和修改先前公开的方法，对拟南芥中20S蛋白酶体进行体外重组测定的详细方案。在此获得20S核心蛋白酶体的主要策略是从26S蛋白酶体中去除19S调节亚基。该协议包括两个主要部分：1）的来自表达表位标记的PAG1稳定的转基因品系20S蛋白酶体亲和纯化，的20S蛋白酶（程序AD）的基本组成部分; 2 ）体外20S蛋白酶体降解测定法（方法E）。我们预计该协议将提供一种简单有效的方法来研究体外20S蛋白酶体降解，并促进植物中蛋白质代谢的研究。
[背景]蛋白质的降解通常是通过真核生物中的蛋白酶体来实现的。整合的26S蛋白酶体由两个亚颗粒组成：一个或两个末端的19S调节颗粒（RP），用作蛋白酶体激活剂；和20S核心蛋白酶体（CP），执行降解过程。大多数真核蛋白被多聚泛素化并导入26S蛋白酶体进行降解。然而，含有固有蛋白质无序已发现的区域直接通过破坏一个由20S蛋白酶的泛素依赖性降解（本日产等人，2014） ...
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| In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1
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Author:
Date:
2015-04-20
[Abstract] microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 ...
[摘要] 微小RNA（miRNA）是小的非编码RNA，其调节真核生物中的大多数（如果不是全部）生物过程。 miRNA最初从初级转录物（pri-miRNA）加工以产生miRNA前体（前-miRNA），其进一步加工成miRNA及其互补链（miRNA/*）。在拟南芥和可能的其他植物中，从pri-miRNA到pre-miRNA和从pre-miRNA到miRNA/*的加工都通过Dicer样1（DCL1）复合物实现。最近，我们证明了从植物中以前的质量的DCL1复合物的分离。我们进一步成功地重建了能够完全重现体内 miRNA生物发生的DCL1切割测定。在这里我们提供DCL1重建测定的详细协议。该方案包括三个主要部分（图1）：1）pri-miRNA和pre-miRNA转录物的制备（方法A-C）; 2）通过免疫沉淀（IP）纯化来自本塞姆氏烟草（本塞姆氏烟草）的重组拟南芥DCL1机器（程序D和E）;和3）使用分离的DCL1复合物（步骤F）在放射性同位素标记的pri-miRNA或pre-miRNA的体外处理。这是我们的愿望，协议是RNAi社区研究机械问题或开发RNA沉默技术的强大工具。
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