Global and local dynamics of the U1A polyadenylation inhibition element (PIE) RNA and PIE RNA-U1A complexes
Global and local dynamics of the U1A polyadenylation inhibition element (PIE) RNA and PIE RNA-U1A complexes
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DOI：
10.1021/bi049117g
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发表时间：
2004-10-26
期刊：
影响因子：
2.9
通讯作者：
Hall, KB
中科院分区：
文献类型：
--
作者：
Clerte, C;Hall, KB
多聚腺苷酸化抑制元件（PIE）RNA的结构和动力学，自由和绑定到U1 A蛋白，已检查使用时间分辨FRET和2-氨基嘌呤（2AP）荧光。该调节RNA位于U1 A前mRNA的3'末端，采用U形结构，在每个弯曲处具有单个U1 A蛋白的结合位点（框1和框2）。RNA的臂末端之间的距离对其三维结构敏感。使用Cy 3/Cy 5 FRET效率监测Mg 2+的结合，我们表明，PIE RNA结合两个Mg 2+离子，这导致其构象的距离分布的限制。使用2AP荧光的局部RNA结构探测显示框2的结构响应于Mg 2+结合而改变，从而试探性地定位离子结合位点。稳态FRET数据显示PIE RNA茎末端之间的距离（R）从游离RNA中的66 A减小到当U1 A的N-末端RNA结合结构域（RBD 1）结合时的58 A，并且当U1 A蛋白结合时减小到53 A。然而，各向异性测量表明Cy 3和Cy 5都堆叠在RNA的末端。为了检查荧光团的受限运动的后果，使用两种不同的运动模型分析FRET数据，然后将其与来自Cy 3/荧光素FRET对的类似数据进行比较。我们得出结论，通过堆叠染料引入距离计算的误差在我们测量的误差范围内。RNA结构的距离分布表明，PIE RNA的臂之间的分子内距离在荧光测量的时间尺度上变化;平均距离取决于蛋白结合，但分布的宽度表明RNA保留结构异质性。
The structure and dynamics of the polyadenylation inhibition element (PIE) RNA, free and bound to the U1A protein, have been examined using time-resolved FRET and 2-aminopurine (2AP) fluorescence. This regulatory RNA, located at the 3' end of the U1A pre-mRNA, adopts a U-shaped structure, with binding sites for a single U1A protein at each bend (box 1 and box 2). The distance between the termini of the arms of the RNA is sensitive to its three-dimensional structure. Using Cy3/Cy5 FRET efficiency to monitor binding of Mg2+, we show that the PIE RNA binds two Mg2+ ions, which results in a restriction of its distance distribution of conformations. Local RNA structure probing using 2AP fluorescence shows that the structure of box 2 changes in response to Mg2+ binding, thus tentatively locating the ion binding sites. Steady-state FRET data show that the distance (R) between the termini of the PIE RNA stems decreases from 66 A in the free RNA, to 58 A when N-terminal RNA binding domains (RBD1) of U1A are bound, and to 53 A when U1A proteins bind. However, anisotropy measurements indicate that both Cy3 and Cy5 stack on the ends of the RNA. To examine the consequences of the restricted motion of the fluorophores, FRET data are analyzed using two different models of motion and then compared to analogous data from the Cy3/fluorescein FRET pair. We conclude that the error introduced into distance calculations by stacking of the dyes is within the error of our measurements. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements; the mean distance is dependent on protein binding, but the breadth of the distributions indicates that the RNA retains structural heterogeneity.