Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744, USA.
RNA. 2010 Dec;16(12):2474-83. doi: 10.1261/rna.2360610. Epub 2010 Oct 27.
In bacteriophage λ, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.
在噬菌体 λ 中，22 个氨基酸肽（称为 N 肽）和茎环 RNA 元件（称为 boxB）之间的相互作用在转录抗终止中起着关键作用。N 肽/boxB 复合物已被广泛研究，并作为理解蛋白质/RNA 识别机制的范例。特别是，超快光谱技术已被应用于监测嵌入 boxB RNA 不同位置的 2-氨基嘌呤的皮秒荧光衰减行为。这些研究提出了一个模型，其中结合的 N 肽在两个状态之间处于动态平衡，肽 C 末端片段要么堆积在（即堆叠状态）要么从（即未堆叠状态）RNA 环上剥离。N 肽/boxB 复合物的功能似乎与堆叠状态的分数相关。在这里，使用定点自旋标记技术研究了 N 肽/boxB 系统，其中 X 波段电子顺磁共振波谱用于监测共价连接到 N 肽不同位置的稳定氮氧自由基的纳秒旋转行为。数据表明，在纳秒范围内，结合的 N 肽的 C 末端片段在复合物中采用多种离散构象。这些构象的特征与提出的堆叠和未堆叠状态一致，并且它们的分布随 N 肽内的突变而变化。这些结果表明，动态双态模型在纳秒范围内仍然有效，并且代表了 N 肽/boxB RNA 复合物中独特的功能模式。它还证明了生物复合物中皮秒和纳秒动力学之间的联系。
