A dimer as a building block in assembling RNA - A hexamer that gears bacterial virus phi29 DNA-translocating machinery
A dimer as a building block in assembling RNA - A hexamer that gears bacterial virus phi29 DNA-translocating machinery
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DOI：
10.1074/jbc.m909662199
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Guo, PX
中科院分区：
文献类型：
--
作者：
Chen, CP;Sheng, ST;Guo, PX
六个RNA （pRNA）分子形成一个六聚体，通过手拉手的相互作用，来齿轮细菌病毒phi29 DNA易位机制。在这里，我们报告了六聚体形成的途径和条件。稳定的pRNA二聚体和三聚体在溶液中组装，从天然凝胶中分离，并通过沉淀分离，为在无蛋白环境下研究RNA二聚体和三聚体提供了一个模型系统。低温原子力显微镜显示单体呈（sic）轮廓，二聚体呈细长形状，三聚体呈三角形。包括亚精胺在内的多种阳离子促进了pRNA的二聚化，而原衣壳结合和DNA包装需要特定的二价阳离子，包括Mg2+、Ca2+和Mn2+。具有互补环的串联和融合pRNA二聚体在DNA包装中都具有活性，而没有互补环的pRNA二聚体则不具有活性。结果表明，二聚体是六聚体的构建单元，构建六聚体的途径为：二聚体->四聚体->六聚体。Hill系数为2.5表明，原衣壳表面存在三个协同结合位点，这两个相互作用的环在招募进入的二聚体中起关键作用，而原衣壳则是六聚体组装的基础。
Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryoatomic force microscopy revealed that monomers displayed a (sic) outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg2+, Ca2+, and Mn2+. Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer, The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid, The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.