The Mammalian Signal Recognition Particle

描述(申请人提供)：信号识别颗粒(SRP)结合翻译核糖体产生的N-末端多肽信号序列，并将该核糖体复合体靶向内质网膜上的受体。多肽以GTP调节的方式转运到膜间隙。哺乳动物SRP由两个结构域组成：Alu和大亚基。大亚基(LS)含有一半的RNA(-150nts)以及SRP19、SRP54和SRP68/SRP72异二聚体蛋白。大亚基执行SRP的大部分功能，包括信号序列识别、GTP结合和水解、受体对接，以及部分延伸停止。该项目的长期目标是以基本和定量的方式了解蛋白质和RNA成分之间的相互作用，以及在哺乳动物SRP大亚基的合作组装和功能中信号肽和核苷酸底物的结合。具体目标是：(1)确定SRP19和SRP54在组装和功能上具有很强协同性的分子基础。(2)了解天然展开的SRP19蛋白与SRP RNA组装的机制。(3)在核苷酸分辨率上定位单个SRP68和SRP72蛋白以及SRP68/72异源二聚体的RNA结合位点，并定量与SRP54和SRP19的协同结合。我们实验室开发了用于确定多组分RNA-蛋白质复合体中平衡结合常数的健壮的单核苷酸解析方法，从而使这些实验的严格分析成为可能。(4)分析完整的SRP大亚基与其两类底物：鸟苷核苷酸和信号肽的相互作用。总体而言，这项工作将解决SRP蛋白质和RNA组件之间的合作相互作用如何发挥作用以进行信号肽识别和核苷酸结合的关键特征。此外，这项工作旨在确定可推广到其他生物和医学上突出的核糖核蛋白复合体的原理。

DESCRIPTION (provided by applicant): The signal recognition particle (SRP) binds the N-terminal polypeptide signal sequence emerging from a translating ribosome and targets this ribosomal complex to a receptor at the endoplasmic reticulum membrane. Polypeptides are translocated into the intermembrane space in a GTP-regulated manner. The mammalian SRP is comprised of two domains: the Alu and large subunits. The large subunit (LS) contains one-half of the RNA (-150 nts) and the SRP19, SRP54, and SRP68/SRP72 heterodimer proteins. The large subunit performs most of the functions of the SRP including signal sequence recognition, GTP binding and hydrolysis, receptor docking, and, in part, elongation arrest. The long term goals of this project are to understand, in a fundamental and quantitative way, the interplay of protein and RNA components and of binding by signal peptide and nucleotide substrates in the cooperative assembly and functioning of the large subunit of the mammalian SRP. Specific aims are: (1) To determine the molecular basis for the strong cooperativity in the assembly and function of SRP19 and SRP54. (2) To understand the mechanism of assembly of the natively unfolded SRP19 protein with the SRP RNA. (3) To map at nucleotide resolution the RNA binding sites for the individual SRP68 and SRP72 proteins and for the SRP68/72 heterodimer and to quantify cooperative binding with SRP54 and SRP19. Rigorous analysis of these experiments is made possible by the development, in our lab, of robust single-nucleotide resolution approaches for determining equilibrium binding constants in multi-component RNA-protein complexes. (4) To analyze interaction of the complete SRP large subunit with its two classes of substrates: guanosine nucleotides and signal peptides. Overall, this work will address key features of how cooperative interactions between SRP protein and RNA components function to carry out signal peptide recognition and nucleotide binding. Moreover, the work is designed to identify principles generalizable to other biologically and medically prominent ribonucleoprotein complexes.