STRUCTURES OF EXOSOME AND SIGNAL RECOGNITION PARTICLE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 我的部分研究集中在一个关键的RNA加工和降解机制，外泌体，它通常被称为RNA的蛋白酶体。最初从引起5.8S rRNA 3'末端加工缺陷的突变中鉴定，外泌体是存在于真核细胞的细胞核和细胞质中的保守的300 - 400 kD 3'至5'核糖核酸外切酶复合物。外泌体由10种蛋白质的核心组成：Rrp 4p、Rrp 40 p至Rrp 46 p、Mtr 3 p和Csl 4p（酵母命名法），它们都是细胞活力所必需的。其中六种是磷酸化核糖核酸酶，其他四种被预测为水解核糖核酸酶。在细胞核中，外泌体是5.8S rRNA的3'端形成和5'-外部转录间隔区降解所必需的;参与小的核和核仁RNA的3'端成熟;并参与低效剪接或低腺苷酸化的前体mRNA的降解。细胞质外泌体参与含有提前终止密码子、缺乏终止密码子或在3'非翻译区附近携带富含AU元件（战神）的mRNA的降解。 为了深入了解外泌体的结构和酶促机制，我提出了确定300 kD，4-蛋白质古细菌外泌体，10-蛋白质真核外泌体和外泌体-RNA底物复合物的晶体结构。长期目标包括确定外泌体-衔接蛋白复合物以研究其调节。在古细菌外泌体复合体的表达、纯化和结晶方面取得了令人兴奋的进展。性能良好的晶体在同步辐射源处衍射X射线至~ 2.4分辨率。需要额外的光束时间来完成Se-MAD定相并对古细菌外泌体复合物进行结构酶学研究。 第二部分研究信号识别粒子（SRP）  介导的蛋白质跨膜或跨膜的共翻译易位。这个重要的细胞过程需要翻译核糖体被SRP膜靶向，SRP是一种在所有三个生命王国中保守的核糖核蛋白复合物。SRP识别从核糖体出现的新生蛋白的疏水信号序列，导致真核生物中的瞬时伸长停滞，并通过与SRP受体（SR）的GTP依赖性相互作用将核糖体靶向至膜。然后核糖体被移交给易位子，在那里蛋白质翻译和易位同时发生。在GTP水解后SRP-SR解离，并且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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.