Complex Riboswitch Structural and Biochemical Analysis

核糖开关是折叠的RNA结构域，其结合特定的代谢物并充当基因表达的调节剂。结合的代谢产物是下游基因的生物合成产物或底物，导致经济的反馈回路机制，用于响应生理需要而改变基因表达。核糖开关由两个功能元件组成：代谢物结合结构域和下游表达平台。代谢物结合诱导的核糖开关构象变化通过改变转录延伸或翻译起始来调节基因表达。虽然核糖开关是普遍存在的，代谢物结合的结构基础和效应促进的构象变化的性质在很大程度上是unknowed.This项目集中在两个复杂的核糖开关：glmS和甘氨酸核糖开关。这两种RNA都表现出超越简单代谢物结合的功能。甘氨酸核糖开关是基因表达的上调因子，并且被发现为两个密切相关的序列的串联重复，其中每个序列能够独立地结合甘氨酸。在串联中，适体协同结合。结果是一个“数字”核糖开关，对甘氨酸浓度非常敏感。这种核糖开关表明，RNA，像蛋白质一样，可以实现小分子的合作变构结合。它是如何做到这一点是未知的。glmS核糖开关作为代谢物依赖性核酶发挥功能。glmS核糖开关响应于代谢物葡糖胺-6-磷酸（Gln 6P），导致glmS基因表达的下调。Gln 6P结合诱导RNA在核糖开关序列的特定残基5'处的自切割。目前还不清楚RNA和Gln 6P是如何相互作用来实现这种活性的。本项目的总体目标是了解这些复杂的核糖核酸调节子的结构和化学基础。该研究将解决以下问题：这些等级如何结合其小分子效应器？glmS RNA如何折叠以产生催化活性位点？Gln 6P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？这两个域是在物理上相互作用，还是在合作上不那么直接？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射线晶体学方法的组合。更广泛的影响：这一项目将继续为学生的研究培训提供一个有利的环境，特别是对传统上在科学领域代表性不足的少数群体的个人。首席研究员担任耶鲁大学分子生物物理学和生物化学系本科生研究主任，每年指导35-50名科学专业学生。他将在本科和研究生阶段教授生物化学，机械酶学和科学逻辑课程。PI将继续担任ASBMB学生校园分会的教师顾问，该分会于去年发起了第一届耶鲁大学本科生科学论坛，该论坛包括学生在学年期间的研究成果的口头和海报展示。有机和大分子化学计划，基因和基因组系统集群以及生物分子系统集群共同支持该项目。

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.