Complex Riboswitch Structural and Biochemical Analysis

核糖开关是折叠的RNA结构域，结合特定的代谢物并作为基因表达的调节剂。结合的代谢物是下游基因的生物合成产物或底物，形成一种经济的反馈循环机制，根据生理需要改变基因表达。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。虽然核糖开关很普遍，但代谢物结合的结构基础和效应物促进构象变化的性质在很大程度上是未知的。本项目主要研究两种复杂的核糖开关：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。甘氨酸核糖开关是基因表达的上调因子，是两个密切相关序列的串联重复序列，每个序列都能独立结合甘氨酸。在串联中，适体协同结合。结果是一个对甘氨酸浓度非常敏感的“数字”核糖开关。这个核糖开关表明，RNA和蛋白质一样，可以实现小分子的合作变构结合。它是如何做到这一点的尚不清楚。glmS核开关作为代谢物依赖的核酶起作用。glmS核开关对代谢物葡萄糖胺-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特定残基5'处的RNA自裂。目前尚不清楚RNA和Gln6P如何相互作用以实现这种活性。这个项目的总体目标是了解这些复杂的核糖体调节剂的结构和化学基础。该研究将解决以下问题：这些分子如何结合它们的小分子效应物？glmS RNA如何折叠以产生催化活性位点？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？这两个领域是否在物理上相互作用，还是合作上不那么直接？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射线晶体学方法的结合。更广泛的影响：该项目将继续为学生的研究培训提供一个培育环境，特别是来自传统上在科学领域代表性不足的少数群体的个人。他担任耶鲁大学分子生物物理和生物化学系本科研究主任，每年指导35-50名科学专业的学生。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学课程。PI将继续担任ASBMB学生校园分会的指导教师，该分会去年发起了第一届耶鲁本科生科学论坛，其中包括学生在学年的研究成果的口头和海报展示。有机与大分子化学项目、基因与基因组系统集群和生物分子系统集群共同支持本项目。

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.