SOLUTION SAXS OF A RNA HELICASE AND ITS COMPLEXES WITH RNA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。DEx(D/H)-box RNA解旋酶是一种多结构域蛋白，在许多不同的情况下负责“伴随”RNA结构，它被认为经历了实质性的构象变化，以催化atp依赖性的靶RNA展开/重折叠。我们研究了一种高亲和力结合23S rRNA特定片段的三结构域解旋酶（即枯草芽孢杆菌的YxiN蛋白），我们建议使用溶液小角度x射线散射来描述该蛋白在RNA结合和驱动解旋酶活性的atp酶循环时所经历的构象变化。已经表达和纯化了全长的YxiN及其亚片段，并解决了YxiN或相近同源物的单个结构域的晶体结构。单个结构域的结构被纳入到全长蛋白及其亚片段的溶液构象的建模中。收集的SAXS数据表明，在没有配体的情况下，YxiN及其2结构域片段的行为就像“弦上的珠子”，结构域之间有柔性的连接体。我们现在希望研究与RNA的相互作用，以确定在什么条件下RNA结合将蛋白质凝聚成解旋酶活性所需的紧凑结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DEx(D/H)-box RNA helicases, which are responsible for "chaperoning" RNA structure in many different contexts, are multi-domain proteins that are thought to undergo substantial conformational changes in order to catalyze ATP-dependent unfolding/refolding of target RNAs. Working with a three-domain helicase that binds specific fragments of 23S rRNA with high affinity (namely, the YxiN protein of Bacillus subtilis), we propose to use solution small angle x-ray scattering to delineate the conformational changes this protein undergoes in response to RNA binding and the ATPase cycle that drives the helicase activity. Both fulllength YxiN and subfragments thereof have been expressed and purified, and crystallographic structures of individual domains of YxiN or close homologs have been solved. Structures of individual domains are being incorporated into modeling the solution conformations of the full-length protein and subfragments thereof. SAXS data have been collected which show that in absence of ligands, YxiN and 2-domain fragments thereof behave like "beads on a string", with flexible linkers between domains. We now wish to examine the interactions with RNA, to determine under what conditions binding of RNA condenses the protein into a compact structure that is required for helicase activity.