STRUCTURES OF EXOSOME AND SIGNAL RECOGNITION PARTICLE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我的部分研究集中在一种关键的RNA加工和降解机制--外切体，它通常被称为RNA的蛋白酶体。外体最初是由导致5.8S rRNA 3‘末端加工缺陷的突变确定的，它是一个保守的300-400kD的3’-5‘外切核糖核酸酶复合体，存在于真核细胞的细胞核和细胞质中。外切体由10种蛋白质组成：Rrp4p、Rrp40p到Rrp46p、mtr3p和Csl4p(酵母命名法)，所有这些都是细胞存活所必需的。其中6个为磷酸化核糖核酸酶，另外4个被预测为水解型核糖核酸酶。在细胞核中，外体对于5.8S rRNA的3‘端形成和5’-外部转录间隔区的降解是必需的，参与了小核和核仁RNA的3‘端成熟，并参与了低度剪接或低腺素化的前mRNAs的降解。胞质外体参与了3‘非翻译区附近含有提前终止密码子、缺少终止密码子或富含AU元素的mRNAs的降解。 为了深入了解Exosome的结构和酶机制，我建议测定300kD，4-preotein in古菌Exosome、10蛋白真核Exosome和Exosome-RNA底物复合体的晶体结构。长期目标包括确定外切体-适配蛋白复合体，以研究其调控。在古菌外体复合体的表达、纯化和结晶方面取得了令人振奋的进展。性能良好的晶体在同步辐射光源下将X射线衍射到~2.4？分辨率。需要额外的束流时间来完成硒相变和对古菌外切体复合体进行结构酶学研究。 我的研究的第二部分集中在信号识别粒子(SRP)介导的蛋白质跨膜或进入膜的共翻译易位。这一重要的细胞过程需要翻译的核糖体被SRP膜靶向，SRP是一种在所有三个生命王国中都保守的核糖核蛋白复合体。SRP识别核糖体产生的新生蛋白的疏水信号序列，导致真核生物的瞬时伸长停止，并通过依赖GTP与SRP受体(SR)的相互作用将核糖体靶向细胞膜。然后，核糖体被移交给转位蛋白，在那里蛋白质翻译和转位同时发生。GTP水解后，SRP-SR解离，SRP循环重新开始。

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. Part of my research focuses on a crucial RNA processing and degradation machinery, the exosome, which has often been called the proteasome for RNA. Initially identified from mutations causing 5.8S rRNA 3' end processing defects, the exosome is a conserved 300 - 400 kD 3'-to-5' exoribonuclease complex present in both the nucleus and the cytoplasm of eukaryotic cells. The exosome consists of a core of ten proteins: Rrp4p, Rrp40p to Rrp46p, Mtr3p, and Csl4p (yeast nomenclature), all are essential for cell viability. Six of them are phosphorolytic RNases; the other four are predicted to be hydrolytic RNases. In the nucleus, the exosome is required for the 3' end formation of 5.8S rRNA and the degradation of the 5'-external transcribed spacer; participated in the 3' end maturation of small nuclear and nucleolar RNAs; and involved in degradation of inefficiently spliced or hypoadenylated pre-mRNAs. The cytoplasmic exosome is involved in the degradation of mRNAs containing premature termination codons, lacking termination codons, or bearing AU-rich elements (AREs) near the 3' untranslated region. To gain insights into the architecture and enzymatic mechanism of the exosome, I have proposed to determine the crystal structures of the 300 kD, 4-preotein archaeal exosome, the 10-protein eukaryotic exosome, and the exosome-RNA substrate complexes. The long term goals include determination of exosome-adaptor protein complexes to investigate its regulation. Exciting progress has been made in expression, purification, and crystallization of the archaeal exosome complex. Well-behaving crystals diffracted X-ray to ~ 2.4 ¿ resolution at synchrotron radiation source. Additional beam time is needed to complete Se-MAD phasing and carry out structural enzymology studies on the archaeal exosome complex. The second part of my research focuses on Signal Recognition Particle (SRP)  mediated co-translational translocation of proteins across or into membranes. This vital cellular process requires the translating ribosome to be membrane-targeted by the SRP, a ribonucleoprotein complex conserved in all three kingdoms of life. SRP recognizes the hydrophobic signal sequence of the nascent protein emerging from the ribosome, resulting in transient elongation arrest in eukaryotes, and targets the ribosome to the membrane via a GTP-dependent interaction with the SRP receptor (SR). The ribosome is then handed over to the translocon, where protein translation and translocation happens simultaneously. The SRP-SR dissociates following GTP hydrolysis and SRP cycle resumes.