Dissection of an RNA Protein Interaction Involve in Repression of Splicing

9513184白色酵母核糖体蛋白L32，RPL 32，与其自身的转录物结合并阻止剪接。 RNA结合靶标可以减少到24个核苷酸，折叠成茎-内环-茎基序，其中环富含嘌呤并通过潜在的G：U对闭合。 先前的实验已经将蛋白质接触的位点定位到环，并且已经完成了一些初步工作来探索蛋白质结合的序列要求。 本文提出的实验直接解决了内环内氢键的问题。 不参与配对的那些碱基的氢键面的化学修饰将通过引物延伸方法检测。 将收集NMR光谱，以检测不与水交换的亚氨基质子。 可见的亚氨基和氨基共振可能涉及氢键。 探索蛋白质结构及其与RNA的界面构成了该提案的第二部分。 目前，该蛋白质只有一级结构是已知的，没有明显的区域与其他蛋白质的RNA结合结构域相关。 首先，将改进纯化方案，并对纯化的蛋白质进行蛋白质分析，将复合物的CD光谱与RNA的CD光谱进行比较，以确定蛋白质结合是否改变RNA的螺旋度。 为了确定哪些蛋白质残基接触RNA，将使用天然和修饰的RNA进行交联实验。 作为这些实验的补充，将对蛋白质进行一种“足迹”实验，以观察蛋白质的哪些部分可以在RNA结合时被保护免于水解。 这些实验将需要一些工作来开发适用于所有RNA蛋白质复合物的实用的通用技术。 在建议的第三部分，也是最后一部分，研究了镇压的机制。 来自一个实验室的证据表明，U1 snRNP与L32 RNA/RPL 32复合物结合，形成不能剪接的三分子复合物。最初的假设是，U1 RNA增强了L32 RNA与RPL 32的结合，并且RPL 32的解离通过与两种RNA的复合而减慢或阻止。教育计划是基于布林莫尔学院前三年的教学。压倒一切的课程理念是设计真正的化学课程，传达当前生物化学的兴奋。 每一次尝试都是为了挑战学生成为积极的，批判性的学习者，他们认为自己是科学事业的参与者。 鉴于布林莫尔学院教育年轻女性的使命，鼓励学生实现他们的科学抱负就显得尤为重要。 对于本科生来说，一整年的研究往往是他们文科教育的高潮，也往往是他们第一次真正独立的研究经历。 这是一个挑战，设计项目，学生可以做成功的实验，同时有助于实验室的进步。解决问题，可视化分子，摩尔，平衡和动力学仍然是学生必须掌握的普通化学的重要组成部分。 因此，入门级教学的挑战是同时保持学生的兴趣，并坚持他们发展未来科学工作所需的技能。 这项研究涉及到对参与调节蛋白质产生的RNA结构的研究。 与DNA形成对称的、细长的双螺旋不同，RNA形成复杂的、回旋的形状。 虽然DNA的所有碱基都参与G-C或A-T配对，但在RNA中，一些碱基配对，而另一些则不配对。 研究的第一部分涉及确定哪些碱基对。 由于其复杂的结构，RNA呈现出多种可被蛋白质识别的特征。 在所研究的系统中，一个小蛋白质与RNA的一部分结合，该部分既有非标准碱基对又有未配对碱基。 将进行实验以确定RNA和蛋白质特征如何有助于这种分子识别事件。 当蛋白质与RNA结合时，其生物学功能被阻断。 将设计模型实验来测试这种蛋白质生产抑制的机制。在教学计划中对高年级学生的生物化学教学和化学导论进行了讨论。 共同的主题是挑战学生学习严格的实验科学，同时分享生物化学当前发展的兴奋。 ***

9513184 White Yeast ribosomal protein L32, RPL32, binds to its own transcript and prevents splicing. The RNA binding target can be reduced to as few at 24 nucleotides folded into a stem-internal loop-stem motif where the loop is purine-rich and is closed by a potential G:U pair. Previous experiments have localized the site of protein contact to the loop and some preliminary work to explore the sequence requirements for protein binding has been completed. The experiments proposed herein directly address the question of hydrogen bonding within the internal loop. Chemical modification of the hydrogen bonding faces of those bases not involved in pairing will be detected by primer extension methods. NMR spectra will be collected to detect imino protons protected from exchange with water. Imino and amino resonances which are visible are probably involved in hydrogen bonds. Probing the protein structure and its interface with RNA makes up the second part of the proposal. At present, only the primary structure of the protein in known and there are no obvious regions related to RNA binding domains of other proteins. At the outset, the purification protocol will be improved and the purified protein will be subjected to analysis of the protein, the CD spectrum of the complex will be compared to that of the RNA to determine whether protein binding changes the helicity of the RNA. To determine which protein residues contact the RNA, crosslinking experiments will be carried out using native and modified RNA. As a complement to these experiments, a type of "footprinting" experiment will be done on the protein to see what portions of the protein may be protected from hydrolysis on RNA binding. These experiments will require some work to develop practical, general techniques which work for all RNA protein complexes. In the third and final portion of the proposal, the mechanism of repression is examined. Evidence from one laboratory suggests that the U1 snRNP binds to the L32RNA/RPL32 complex to form a termolecular complex incapable of splicing. The initial hypothesis is that the U1 RNA strengthens the binding of L32 RNA to RPL32 and that the dissociation of RPL32 is slowed or prevented by complexation with the two RNAs. The Education Plan is based on the first three years of teaching at Bryn Mawr College. The overriding curricular philosophy is to devise genuine chemistry courses which convey the current excitement in biochemistry. Every attempt is made to challenge the students to become active, critical learners who see themselves as participants in the scientific enterprise. Given Bryn Mawr's mission to educate young women, it is particularly important to encourage its students to realize their scientific ambitions. For undergraduates, a full year of research is often the culmination of their liberal arts education and is frequently their first truly independent research experience. It is a challenge to design projects where students can do successful experiments while contributing to the progress of the laboratory. Problem solving, visualizing molecules, moles, equilibria, and kinetics are still vital parts of General Chemistry which students must master. The challenge of teaching at the introductory level is thus to simultaneously hold the students' interest and insist that they develop the skills needed for future work in science. %%% This research involves the study of the structure of an RNA involved in regulating the production of a protein. Unlike DNA, which forms a symmetric, elongated double helix, RNA forms a complex, convoluted shape. While all of DNA's bases are involved in either G-C or A-T pairs, in RNA some bases are paired but others are not. The first portion of the research involves determining which bases pair. Due to its complicated structure, RNA presents a variety of features which could be recognized by a protein. In the system under study, a small protein binds to a portion of an RNA which has both non-standard base pairs and unpaired bas es. Experiments will be done to define how the RNA and protein features contribute to this molecular recognition event. When the protein is bound to the RNA, its biological function is blocked. Model experiments will be designed to test mechanisms for this repression of protein production. The teaching of biological chemistry to advanced students and introductory chemistry is discussed in the education plan. The common theme is to challenge the students to learn a rigorous experimental science while sharing in the excitement of current developments in biochemistry. ***