CHARACTERIZATION OF LIGAND RECOGNITION OF A METABOLITE-RESPONSIVE RIBOZYME

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。细胞的代谢状态通常通过蛋白质介导的转录或翻译控制机制来影响基因的表达。然而，最近的研究表明，信使RNA(MRNAs)可以通过被称为核糖开关的固有结构域直接感知和响应特定的代谢物。在代谢产物存在的情况下，核糖切换域的结构变化可以导致转录终止或翻译抑制。最近，一种新的催化核糖开关被发现，它通过对新生RNA的自我切割来对细胞代谢物浓度做出反应，从而实施遗传控制。依赖代谢物的核酶位于许多革兰氏阳性细菌GLMS基因的5‘非翻译区，它催化内部的磷酸酯转移反应，导致该mRNA的切割和失活。核酶选择性地识别氨基葡萄糖-6-磷酸，并被其1000倍激活，氨基葡萄糖-6-磷酸是GLMS酶的代谢产物，也是细菌细胞壁的重要组成部分。我们感兴趣的是了解GLMS核糖开关识别和催化配体的分子基础。为了解决这个问题，我们测量了核酶/核糖开关对一组相关但不同的配体，如丝氨醇、葡萄糖和氨基葡萄糖等的自裂解速度。我们已经确定核酶与天然配体中的胺和磷酸盐有重要的接触。我们有兴趣确定这两个官能团是如何与全长GLMS核酶相互作用的，以便充分了解它在催化中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The metabolic state of a cell typically affects gene expression through protein-mediated mechanisms of transcriptional or translational control. Yet recent studies have shown that messenger RNAs (mRNAs) can directly sense and respond to specific metabolites through intrinsic domains termed riboswitches. In the presence of metabolites, structural changes in riboswitch domains can result in either transcriptional termination or translational repression. Recently a novel catalytic riboswitch has been discovered that exerts genetic control through self-cleavage of the nascent RNA in response to cellular metabolite concentration. The metabolite-dependent ribozyme resides in the 5'-untranslated region of the glmS mRNA of numerous Gram-positive bacteria and it catalyzes an internal phosphoester transfer reaction that results in cleavage and inactivation of the mRNA. The ribozyme selectively recognizes and is 1000-fold activated by glucosamine-6-phosphate, the metabolic product of the GlmS enzyme, and an important component of bacterial cell walls. We are interested in understanding the molecular basis of ligand recognition and catalysis by the glmS riboswitch. To address this we measured the rate of self-cleavage of the ribozyme/riboswitch in response to a panel of related but distinct ligands, serinol, glucose and glucosamine, to name a few. We have determined that the ribozyme makes important contacts to the amine and phosphate in the natural ligand. We are interested in determining how these two functional groups are interacting with the full-length glmS ribozyme in order to fully understand its role in catalysis.