Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.
Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.
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通过蛋白激酶 PKR 的双链 RNA 结合结构域拉直凸出的 RNA。
DOI：
10.1073/pnas.011355798
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发表时间：
2000
影响因子：
11.1
通讯作者：
Bevilacqua,PC
中科院分区：
文献类型：
--
作者：
Zheng,X;Bevilacqua,PC
人干扰素诱导蛋白激酶 PKR 是一种抗病毒剂，由长链双链 (ds)RNA 激活。 PKR 具有 N 端 dsRNA 结合结构域，其中包含 dsRNA 结合基序的两个串联拷贝，并以非序列特异性方式与 dsRNA 相互作用。令人惊讶的是，PKR 可以受到某些含有非 Watson-Crick 特征的病毒和细胞 RNA 的调节。 我们发现螺旋中部含有凸起的 RNA 可以与 p20 结合，p20 是一种含有 dsRNA 结合结构域的 C 端截短的 PKR。 众所周知，凸起会通过弯曲螺旋轴来改变 RNA 的整体几何形状。因此，我们研究了 PKR 结合引起的凸出 RNA 的构象变化。构建了 66 聚体 DNA-RNA(+/- A3bulge)-DNA 嵌合体并与互补 RNA 链退火。这种双链体迫使蛋白质在中间结合。使用具有由 DNA-DNA(+/-A3bulge)-RNA 组成的顶链的 66 聚体双链体作为对照。 RNA-蛋白质复合物之间的凝胶迁移率变化与蛋白质结合时凸出的RNA的拉直一致。此外，p20 与凸出 RNA 结合的 van't Hoff 分析揭示了相对于与直链 dsRNA 的结合有利的 ΔΔH° 和不利的 ΔΔS°。这些热力学参数与 RNA 伸直的最近邻分析的预测非常一致，并支持一个模型，其中凸起侧翼的螺旋连接点堆叠在蛋白质结合上。 dsRNA 结合基序蛋白识别和拉直弯曲 RNA 的能力对于调节体内 RNA 的拓扑结构具有重要意义。
The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson–Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/− A3bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/−A3bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable ΔΔH° and an unfavorable ΔΔS° relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAsin vivo.