STRUCTURAL STUDIES OF MRNA METABOLISM & SUMO PROTEIN MODIFICATION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。信使核糖核酸成熟和衰退。细胞内信使核糖核酸的稳定性和寿命取决于前信使核糖核酸的加工和衰变途径。我们研究了与RNA封顶相关的早期加工事件，包括真核生物封顶新生mRNA链所需的催化步骤。该基因的5？三磷酸(Pppn)被RNA三磷酸酶切割，生成5？二磷酸(PPN)的mRNA分子。在将GMP从GTP转移到RNA的5？二磷酸末端(GpppN)的反应中，该反应产物是RNA鸟苷酸转移酶的底物。鸟苷酸盐被RNA(鸟嘌呤-7)甲基转移酶甲基化，形成功能性的m7GpppN帽。每一种帽子形成活动都是细胞生长所必需的。我们正在研究几种酶相互作用的结构基础，以及与RNA和寡核苷酸化合物的复合体，以及与来自RNA聚合酶II的磷酸化CTD的复合体。RNA衰变在RNA代谢中也起着重要的作用，我们从事的研究旨在阐明控制几种衰变途径的调控机制。相扑。小的泛素样修饰物SUMO调节真核生物的核运输、应激反应和信号转导，这一过程对酵母的细胞周期进程至关重要。与泛素修饰类似，相扑结合发生在赖氨酸残基上，并由相扑激活酶E1、相扑结合酶E2、相扑结合酶、E3样结合辅因子以及催化相扑加工和去共轭的蛋白酶催化。相扑修饰似乎并不针对降解的蛋白质，而是通过改变细胞定位、生化激活或通过保护泛素依赖的降解来改变目标蛋白质的功能。我们已经从结构上描述了这个系统的几个组成部分，既有单独的，也有相互复杂的。我们目前正在表征E1、E2、E3、相扑与各种底物和辅因子之间额外络合物的结构基础。

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. mRNA maturation and decay. The stability and lifetime of cellular mRNA depends on pre-mRNA processing and decay pathways. We study early processing events associated with RNA capping including the catalytic steps that are required in eukaryotic organisms to cap the nascent mRNA chain. The 5¿ triphosphate (pppN) of the mRNA is cleaved by RNA triphosphatase to produce a 5¿ diphosphate (ppN) mRNA molecule. This reaction product is a substrate for RNA guanylyltransferase in a reaction that transfers GMP from GTP to the 5¿ diphosphate end of the RNA (GpppN). The guanylate is methylated by RNA (guanine-7) methyltransferase to form the functional m7GpppN cap. Each of the cap-forming activities is essential for cell growth. We are characterizing the structural basis for several enzymes in complex with each other, in complex with RNA and oligonucleotide compounds, and in complex with phosphorylated CTD from RNA polymerase II. RNA decay also plays an important role in RNA metabolism, and we are engaged in studies aimed at elucidating regulatory mechanisms controlling several decay pathways. SUMO. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes, a process that is essential for cell cycle progression in yeast. Analogous to ubiquitin modification, SUMO conjugation occurs on lysine residues and is catalyzed by E1, the SUMO activating enzyme, E2, the SUMO conjugation enzyme, E3-like conjugation cofactors, and proteases that catalyze SUMO processing and deconjugation. SUMO modification does not appear to target proteins for degradation, but rather alters the target protein function through changes in cellular localization, biochemical activation, or through protection from ubiquitin-dependent degradation. We have structurally characterized several components of this system, both alone and in complex with each other. We are currently characterizing the structural basis for additional complexes between E1, E2, E3, SUMO and various substrates and cofactors.