Franco-Echevarría Elsa, González-Polo Noelia, Zorrilla Silvia, Martínez-Lumbreras Santiago, Santiveri Clara M, Campos-Olivas Ramón, Sánchez Mar, Calvo Olga, González Beatriz, Pérez-Cañadillas José Manuel
Departament of Crystallography and Structural Biology, Institute of Physical-Chemistry "Rocasolano", CSIC, C/ Serrano 119, 28006 Madrid, Spain.
Instituto de Biología Funcional y Genómica, IBFG-CSIC, Universidad de Salamanca.
Nucleic Acids Res. 2017 Sep 29;45(17):10293-10305. doi: 10.1093/nar/gkx685.
Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.
在酵母中，非编码RNA的转录终止由三种RNA结合蛋白组成的复合物调控：Nrd1、Nab3和Sen1。Nrd1在此过程中起核心作用，它与RNA聚合酶II的Rbp1、TRAMP的Trf4以及GUAA/G终止序列相互作用。我们缺乏这些结合事件中最后一项的结构数据。我们确定了Nrd1 RNA结合结构域及其与三种含GUAA的RNA形成的复合物的结构，表征了RNA结合能，并在体内测试了合理设计的突变体。Nrd1结构显示一个RNA识别基序（RRM）结构域与第二个α/β结构域融合，我们将其命名为分裂结构域（SD），因为它由RRM两侧的两个不连续片段组成。GUAA与这两个结构域以及夹在两个堆积腺嘌呤和SD之间的水分子口袋相互作用。全面的结合研究首次证明，Nrd1对GUAA的偏好略高于GUAG，遗传和功能研究表明，Nrd1 RNA结合结构域可能在非编码RNA转录终止中发挥进一步作用。
