In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
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Author:
Date:
2018-01-05
[Abstract] RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.
[摘要] 真核生物中的RNA依赖性RNA聚合酶(RdRP)将单链RNA转化为双链RNA,从而扩增在调节发育,维持基因组完整性和抗病毒免疫方面起关键作用的小干扰RNA。 在此,我们描述了使用通过昆虫表达系统制备的重组拟南芥RDR6的体外RdRP测定的方法。 通过使用这种经典的生物化学分析,我们发现RDR6有一个强大的模板偏好RNAs缺乏poly(A)尾巴。 这个简单的方法将适用于拟南芥属和其他生物体中的其他RdRPs。
【背景】已经在所有真核生物王国 - 植物,真菌,原生动物和动物中发现RNA依赖性RNA聚合酶(RdRP)基因(Zong等人,2009)。它们将单链RNA(ssRNA)转化为双链RNA(dsRNA),从而扩增在各种生物过程中发挥关键作用的小干扰RNA(siRNA),包括调节发育(Peragine等人, ,2004; Li等人,2005),维持基因组完整性(Volpe等人,2002; Xie等人,2004年, )和抗病毒免疫性(Mourrain等人,2000; Yu等人,2003; Garcia-Ruiz等人,2010; Wang ,2010)。除了这种RdRP活性之外,RdRP还具有称为末端核苷酸转移酶(TNTase)活性的另一种酶活性(Curaba和Chen,2008; ...
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Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
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Author:
Date:
2017-08-20
[Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough ...
[摘要] 我们已经开发了产生组氨酸激酶DesK及其同源反应调节物DesR的位点特异性变体的方案,有助于捕获蛋白质的不同信号状态。两个合作伙伴在大肠杆菌中的共表达,确保调节剂过量,对于DesK:DesR复合物的可溶性生产和进一步纯化是至关重要的。通过使用分子置换的X射线晶体学解决了捕获在磷酸转移酶和磷酸酶反应步骤中的复合物的3D结构。该解决方案不是微不足道的,我们发现在用作搜索探针的硅片生成的模型中,有助于将大部分复合物放置在不对称单元中。电子密度图就足够清楚了,可以进行人工建模,获得完整的原子模型。这些方法有助于解决细菌信号领域的主要挑战,即获得稳定的激酶:调节复合物,具有不同的构象状态,适用于高分辨率晶体学研究。 【背景】关于细菌信号复合物,特别是双组分系统(TCS)的结构信息仍然很少(Casino et al。,2009; Gao and Stock,2009)。 TCS包含几乎所有细菌中的感觉组氨酸激酶(HK)和响应调节剂(RR)配偶体,它们允许细胞感知环境并通过适应性反应相应地反应。尽管在信号传输中这种切换机制的重要性(Trajtenberg等,2016),结构信息对于采用不同功能状态的TCS复合体甚至更为有限。我们研究了DesK-DesR途径(de Mendoza,2014),一种来自枯草芽孢杆菌的TCS,其参与调节细胞膜组成以适应降低双层流动性的线索,如冷休克。 ...
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