CHARACTERIZATION OF LIGAND RECOGNITION OF A METABOLITE-RESPONSIVE RIBOZYME

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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产物。 在代谢物的存在下，核糖开关结构域的结构变化可以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，其通过响应细胞代谢物浓度的新生RNA的自切割来施加遗传控制。 代谢物依赖性核酶位于许多革兰氏阳性细菌glmS mRNA的5 '-非翻译区，催化导致mRNA裂解和失活的内部磷酸酯转移反应。 核酶选择性地识别葡萄糖胺-6-磷酸并被葡萄糖胺-6-磷酸激活1000倍，葡萄糖胺-6-磷酸是GlmS酶的代谢产物，并且是细菌细胞壁的重要组分。 我们有兴趣了解的配体识别和催化的glmS核糖开关的分子基础。 为了解决这一问题，我们测量了核酶/核糖开关响应于一组相关但不同的配体（丝氨醇、葡萄糖和葡糖胺，仅举几例）的自切割速率。 我们已经确定，核酶使重要的天然配体中的胺和磷酸盐的接触。 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.