CHARACTERIZATION OF LIGAND RECOGNITION OF A METABOLITE-RESPONSIVE RIBOZYME

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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影响基因表达。然而，最近的研究表明，信使rna （mrna）可以通过称为核糖开关的内在结构域直接感知和响应特定的代谢物。在代谢物存在的情况下，核糖开关结构域的结构变化可能导致转录终止或翻译抑制。最近发现了一种新的催化核糖开关，它通过新生RNA的自裂解来响应细胞代谢物浓度，从而施加遗传控制。这种依赖代谢物的核酶存在于许多革兰氏阳性细菌glmS mRNA的5'-未翻译区，它催化内部磷酸酯转移反应，导致mRNA的裂解和失活。该核酶选择性识别并被葡萄糖胺-6-磷酸激活1000倍，葡萄糖胺-6-磷酸是GlmS酶的代谢产物，是细菌细胞壁的重要成分。我们感兴趣的是了解配体识别和glmS核开关催化的分子基础。为了解决这个问题，我们测量了核酶/核开关对一组相关但不同的配体（丝氨酸醇、葡萄糖和氨基葡萄糖）的自裂率。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如何与全长glmS核酶相互作用，以充分了解其在催化中的作用。

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.