Chaperones & Degradosomes: Modulation of RNA Activity

描述（由申请人提供）：在体内，存在许多通过调节或修饰RNA结构来精确调节RNA活性的机制。在RNA修饰的广泛领域内，我们正在对两个系统进行结构和生物物理研究。第一个是RNA分子伴侣家族，称为“DEX（D/H）-盒解旋酶”，其催化RNA结构在ATP依赖性活性中的重排。这些蛋白质在大小（范围从大约400到超过1200个残基）和功能（范围从参与RNA剪接和核糖体组装到RNA降解）方面是多样的;它们共享保守的大约400个残基的解旋酶“核心片段”，并且通过赋予特异性的不同外围结构域来区分。解旋酶核心的晶体结构已经确定，揭示了这个蛋白质家族如何适应解旋酶结构的更广泛的图景。这一领域未来工作的重点是：（a）解旋酶及其与RNA的复合物的整体构象是什么？（B）ATP驱动的解旋酶活性和特异性RNA底物识别的结构基础是什么？（c）“特异性结构域”的靶向募集机制有多普遍？这些问题正在解决与蛋白质的一个亚家族，参与核糖体的生物合成，集中体现在E。coli DbpA和B.枯草芽孢杆菌YxiN，其以高亲和力结合特异性RNA靶序列。长期目标是使用溶液小角X射线散射探测解旋酶的溶液构象，使用X射线晶体学解决与特定RNA寡核苷酸复合的解旋酶或其亚片段的结构，并通过用U1 A RNA结合模块替换特异性结构域来测试靶招募机制。 正在研究的第二个系统是核糖核酸内切酶RNase E，它既是原核RNA降解体的基本酶组分，也是核糖体和信使RNA的加工酶。RNase E是一种大蛋白，具有负责核酸酶活性的催化结构域和将伴侣蛋白募集到降解体的支架结构域。通过系统的结构基因组学方法，RNase E的催化片段已被定义并结晶;晶体的分辨率为~3.2 A;结构将使用标准方法解析。通过诱变、酶学分析和晶体学研究的结合，阐明了RNase E的体外催化机制和底物结合特异性。将与Stanley Cohen实验室合作，确定体外活性改变对体内活性和RNase E调节的影响。

DESCRIPTION (provided by applicant): In vivo, there are numerous mechanisms whereby RNA activities are precisely regulated by modulation or modification of RNA structures. Within the broad field of RNA modification, we are pursuing structural and biophysical studies on two systems. The first is a family of RNA chaperones known as the "DEx(D/H)-box helicases" which catalyze rearrangement of RNA structures in ATP-dependent activities. These proteins are diverse with respect to size (ranging from approximately 400 to over 1200 residues) and function (ranging from participation in RNA splicing and ribosome assembly to RNA degradation); they share a conserved, approximately 400 residue helicase "core fragment", and are distinguished by different peripheral domains that confer specificity. The crystallographic structure of the helicase core has been determined, revealing how this family of proteins fits into the broader picture of helicase architecture. The foci of future work in this area are (a) what are the global conformations of the helicases and their complexes with RNA that are relevant to helicase activity; (b) what is the structural basis for ATP-driven helicase activity and specific RNA substrate recognition; and (c) how general is the mechanism of target recruitment by a "specificity domain"? These questions are being addressed with a subfamily of the proteins that are involved in ribosome biogenesis, epitomized by E. coli DbpA, and B. subtilis YxiN, which bind specific RNA target sequences with high affinity. The long-term goals are to probe the solution conformations of the helicase using solution small-angle x-ray scattering, solve the structure of the helicase or subfragments, thereof, complexed to specific RNA oligonucleotides using x-ray crystallography, and to test the target recruiting mechanism by replacing the specificity domain with a U1A RNA binding module. The second system being studied is the endoribonuclease RNase E, which is both an essential enzymatic component of the prokaryotic RNA degradosome, and a processing enzyme for ribosomal and messenger RNA. RNase E is a large protein with both a catalytic domain that is responsible for the nuclease activity, and a scaffolding domain that recruits partner proteins to the degradosome. Through a systematic structural genomics approach, a catalytic fragment of RNase E has been defined and crystallized; the crystals diffract to ~3.2 A resolution; the structure will be solved using standard methods. The in vitro catalytic mechanism and substrate binding specificity of RNase E will be elucidated through a combination of mutagenesis, enzymatic assay and crystallographic studies. The effects of alteration of in vitro activity on in vivo activity and regulation of RNase E will be determined, in collaboration with the Stanley Cohen lab.