POST-TRANSLATIONAL PROTEIN MODIFICATION AND RNA PROCESSING AND DECAY

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。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它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， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 mRNA加帽和SUMO蛋白修饰的结构研究。mRNA加帽。 在所有真核生物中，需要三个催化步骤来适当地给新生mRNA链加帽。mRNA的5'三磷酸（pppN）被RNA三磷酸酶在？位置切割，产生5'二磷酸（ppN）mRNA分子。在将GMP从GTP转移到产生GpppN的RNA的5'二磷酸末端的反应中，该反应的产物是RNA鸟苷酰转移酶的底物。帽鸟苷酸然后被RNA（鸟嘌呤-7）甲基转移酶甲基化以形成功能性m7 GpppN结构。每一个帽形成活动是必不可少的芽殖酵母细胞生长。我们正在表征这些酶中的几种相互复合、与各种RNA和寡核苷酸化合物复合以及与来自RNA聚合酶II的磷酸化CTD复合的结构基础。相扑。已知小泛素样修饰物SUMO调节真核生物中的核转运、应激反应和信号转导，这是酵母中细胞周期进展所必需的过程。与泛素修饰类似，SUMO缀合发生在赖氨酸残基上，并由E1（SUMO活化酶）、E2（SUMO缀合酶）、E3样缀合辅因子和催化SUMO加工和解缀合的蛋白酶催化。SUMO修饰似乎并不靶向降解蛋白，而是通过细胞定位、生物化学活化或通过保护免受泛素依赖性降解来改变靶蛋白功能。我们已经在结构上描述了这个系统的几个组成部分，无论是单独的还是相互复杂的。我们目前正在表征E1，E2，E3，SUMO和各种底物和辅因子之间的其他复合物的结构基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. 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. Structural studies of mRNA capping & SUMO protein modification. mRNA capping. Three catalytic steps are required in all eukaryotic organisms to properly cap the nascent mRNA chain. The 5' triphosphate (pppN) of the mRNA is cleaved by RNA triphosphatase at the ¿ position to produce a 5' diphosphate (ppN) mRNA molecule. The product of this reaction is substrate for RNA guanylyltransferase in a reaction that transfers GMP from GTP to the 5' diphosphate end of the RNA producing GpppN. The cap guanylate is then methylated by RNA (guanine-7) methyltransferase to form the functional m7GpppN structure. Each of the cap- forming activities is essential for cell growth in budding yeast. We are characterizing the structural basis for several of these enzymes in complex with each other, in complex with various RNA and oligonucleotide compounds, and in complex with the phosphorylated CTD from RNA polymerase II. SUMO. The small ubiquitin-like modifier SUMO is known to regulate 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.