STRUCTURAL INVESTIGATIONS OF RIBOZYMES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们一直在研究RNA酶催化的结构方面。我们目前的工作集中在I组内含子和代谢物反应性核酶GLMS上。GLMS核酶被小糖氨基葡萄糖-6-磷酸(GlcN6P)激活。这种核酶被归类为核糖开关，因为切割事件导致许多革兰氏阳性细菌中GlcN6P的生成下调。GLMS核酶的RNA结构促进自身切割的确切化学机制尚不清楚。这种核酶是利用催化金属离子，如I族内含子，还是利用一种或多种杂环碱催化普通酸或普通碱，如HDV核酶？GlcN6P是引起构象变化以促进激活的RNA折叠，还是它的一个官能团，如氨基，直接参与了化学反应？如果是后者，这将是第一个利用小分子辅因子来促进化学作用的RNA的例子。有趣的是，GlcN6P的氨基是活性所必需的，并且这种胺的PKA随核酶的PKA而变化。这种核酶的晶体结构将为这些生化问题提供重要的洞察力。它还将为设计和解释这种RNA的所有后续生化研究提供一个结构框架。

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. We have been investigating structural aspects of catalysis by RNA enzymes. Our current work focuses on the group I intron and the metabolite responsive ribozyme, GlmS. The GlmS ribozyme is activated by the small sugar, glucosamine-6-phosphate (GlcN6P). This ribozyme is classified as a riboswitch, as the cleavage event results in the down regulation of GlcN6P production in many Gram positive bacteria. The precise chemical mechanism by which the RNA structure of the GlmS ribozyme promotes self cleavage is unknown. Does this nucleolytic ribozyme utilize catalytic metal ions, such as with the group I intron, or does it employ one or more of the heterocyclic bases for general acid or general base catalysis, such as the HDV ribozyme? Does the GlcN6P induce a conformational change to promote an activated RNA fold, or does one of its functional groups, such as the amino group, participate directly in the chemical reaction? If the latter is the case, it would be the first example of an RNA that utilizes a small molecule cofactor to promote chemistry. Interestingly, the amino group of GlcN6P is absolutely required for activity and the pKa of this amine varies with the pKa of the ribozyme. The crystal structure of this ribozyme would provide significant insight into these biochemical questions. It would also provide a structural framework for designing and interpreting all subsequent biochemical studies of this RNA.