Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA.
J Am Chem Soc. 2012 Feb 8;134(5):2644-52. doi: 10.1021/ja2093647. Epub 2012 Jan 27.
The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.
噬菌体 phi29 基因组 DNA 凝结成其预先形成的衣壳需要 DNA 包装马达，这是已知最强的生物马达。包装马达是一种复杂的环形蛋白质/RNA 复合物，其功能需要一种称为包装 RNA（pRNA）的 RNA 成分。目前对 pRNA 的结构信息有限，这阻碍了对马达功能的研究。在这里，我们使用定点自旋标记来绘制连接马达 ATP 酶和衣壳的结合位点的 pRNA 三链结的构象。这些研究是在 pRNA 二聚体上进行的，pRNA 二聚体是最简单的环形 pRNA 复合物，是马达组装过程中的功能中间体。使用不依赖核苷酸的标记方案，将稳定的氮氧自由基附着到八个特定的 pRNA 位点上，而不会干扰 RNA 折叠和二聚体形成，并使用双电子电子共振光谱法测量跨越三链结的总共 17 个内氮氧自由基距离。测量的距离，加上空间化学约束，用于从 650 亿个池中选择 3662 个可行的三链结模型。结果表明，可行模型之间存在相似的构象，其中两个螺旋（H(T)和 H(L)）采用锐角弯曲。这与最近报道的 pRNA 四聚体晶体结构形成对比，其中 H(T)和 H(L)线性堆叠在一起。这些研究建立了一种用于绘制复杂 RNA 分子全局结构的新方法，并提供了有关 pRNA 构象的信息，这有助于 phi29 包装马达的研究以及基于 pRNA 的纳米医学和纳米材料的发展。
