Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Mexico.
Biochimie. 2013 Nov;95(11):2034-41. doi: 10.1016/j.biochi.2013.07.022. Epub 2013 Aug 6.
We report an analysis in vivo of the RNA degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E-PNPase (polynucleotide phosphorylase), and RNase E-Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present evidence for in vivo proximity of Enolase-PNPase and Enolase-RhlB. The data support a model for the RNA degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.
我们报告了大肠杆菌中 RNA 降解体组装的体内分析。通过荧光显微镜成像和荧光能量转移（FRET）测量，我们提供了证据证明体内 RNase E-PNPase（多核苷酸磷酸化酶）和 RNase E-烯醇酶之间存在成对相互作用。在具有基因组编码的 RNase E 的突变株中，这些相互作用不存在，该突变株缺乏羧基末端一半，支持羧基末端结构域作为降解体支架的作用。我们还提供了 Enolase-PNPase 和 Enolase-RhlB 体内接近的证据。这些数据支持 RNA 降解体（RNAD）的模型，其中 RNase E 的羧基末端与 PNPase 接近，与烯醇酶更远，与 RhlB 解旋酶超过 10nm。我们的测量是在具有单拷贝染色体融合的菌株中进行的，这些菌株的 RNAD 酶与荧光蛋白融合，允许在不同的生长和应激条件下测量不同蛋白质的表达。
