Complex Riboswitch Structural and Biochemical Analysis

核糖开关是折叠的RNA结构域，它结合特定的代谢物，并作为基因表达的调节器。结合的代谢物是下游基因的生物合成产物或底物，导致了一种经济的反馈循环机制，以改变基因表达以响应生理需求。核糖开关由两个功能元件组成：代谢物结合域和下游表达平台。代谢产物结合引起的核糖开关的构象变化通过改变转录延伸或翻译起始来调节基因的表达。虽然核糖开关很普遍，但代谢物结合的结构基础和效应器促进的构象变化的性质在很大程度上尚不清楚。本项目重点研究两个复杂的核糖开关：GLMS和甘氨酸核糖开关。这两种RNA的功能都超越了简单的代谢物结合。甘氨酸核糖开关是一种基因表达上调因子，被发现是两个密切相关的序列的串联重复，每个序列都能够独立地与甘氨酸结合。在串联中，适配子协同结合。结果是一种“数字”核糖开关，它对甘氨酸浓度非常敏感。这种核糖开关表明，RNA和蛋白质一样，可以实现小分子的协同变构结合。它是如何做到这一点的还不得而知。GLMS核糖开关作为代谢物依赖的核酶发挥作用。GLMS核糖开关对代谢产物氨基葡萄糖-6-磷酸(Gln6P)有反应，导致GLMS基因表达下调。Gln6P结合诱导核糖开关序列的特定残基5‘处的RNA自我切割。目前尚不清楚RNA和Gln6P是如何相互作用实现这种活性的。这个项目的总体目标是了解这些复杂的核转录调节因子的结构和化学基础。这项研究将解决以下问题：这些序列是如何结合它们的小分子效应器的？GLMS RNA如何折叠以创建催化活性部位？Gln6P是简单地诱导构象变化来激活RNA，还是它的一个官能团直接参与了化学反应？甘氨酸核糖开关如何实现两个甘氨酸分子的协同结合？这两个领域是在物理上相互作用，还是在合作方面不那么直接？为了回答这些问题，该项目将使用有机合成、RNA生物化学和X射线结晶学方法的组合。更广泛的影响：该项目将继续为学生，特别是传统上在科学界代表性较低的少数群体的个人的研究培训提供一个培养环境。这位首席研究员担任耶鲁大学分子生物物理和生物化学系本科生研究主任，每年指导35-50名理科专业的学生。他将在本科生和研究生阶段教授生物化学、机械酶学和科学逻辑课程。该协会将继续担任ASBMB学生校园分会的教师顾问，该分会去年发起了首届耶鲁大学本科生科学论坛，其中包括学生在学年中的研究成果的口头和海报演示。有机化学和高分子化学项目、基因和基因组系统集群以及生物分子系统集群共同支持这一项目。

Riboswitches are folded RNA domains that bind specific metabolites and act as regulators of gene expression. The bound metabolites are the biosynthetic products or substrates of the downstream genes, resulting in an economical feedback loop mechanism for altering gene expression in response to physiological needs. Riboswitches are composed of two functional elements: a metabolite binding domain and a downstream expression platform. Conformational changes in the riboswitch induced by metabolite binding result in modulation of gene expression by altering transcriptional elongation or translational initiation. While riboswitches are prevalent, the structural basis of metabolite binding and the nature of the effector promoted conformational changes are largely unknown.This project focuses on two complex riboswitches: the glmS and the glycine riboswitch. Both of these RNAs demonstrate functions that go beyond simple metabolite binding. The glycine riboswitch is an upregulator of gene expression and is found as a tandem repeat of two closely related sequences, each of which is able to independently bind glycine. In tandem the aptamers bind cooperatively. The result is a "digital" riboswitch that is exquisitely sensitive to the glycine concentration. This riboswitch demonstrates that RNA, like protein, can achieve cooperative allosteric binding of a small molecule. How it does this is unknown. The glmS riboswitch functions as a metabolite dependent ribozyme. The glmS riboswitch is responsive to the metabolite glucosamine-6-phosphate (Gln6P) resulting in down regulation of glmS gene expression. Gln6P binding induces self-cleavage of the RNA at a specific residue 5' of the riboswitch sequence. It is unclear how the RNA and Gln6P interact to achieve such activity.The overall goal of this project is to understand the structural and chemical basis of these complex riboregulators. The research will address the following questions: How do these ranks bind their small molecule effectors? How does the glmS RNA fold to create an active site for catalysis? Does the Gln6P simply induce a conformational change to activate the RNA, or does one of its functional groups participate directly in the chemical reaction? How does the glycine riboswitch achieve cooperative binding of two glycine molecules? Do the two domains physically interact with each other, or is the cooperatively less direct? To answer these questions, the project will use a combination of organic synthesis, RNA biochemistry and X-ray crystallography methods. Broader Impacts: This project will continue to provide a nurturing environment for research training of students, particularly of individuals from minority groups traditionally underrepresented in the sciences. The Principle Investigator serves as the Director of Undergraduate Studies in the Molecular Biophysics and Biochemistry Department at Yale University, where he mentors 35-50 science majors per year. He will teach courses in Biochemistry, Mechanistic Enzymology and Scientific Logic at the undergraduate and graduate levels. The PI will continue to serve as the faculty advisor to the student campus chapter of the ASBMB, which last year initiated the first annual Yale Undergraduate Science Forum that included oral and poster presentations of the student's research accomplishments during the academic year.Organic and Macromolecular Chemistry Program, Genes and Genome Systems Cluster, and Biomolecular Systems Cluster support this project jointly.