STRUCTURAL INVESTIGATIONS OF RIBOZYMES

该子项目是利用 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，不一定是研究者的机构。 我们一直在研究RNA酶催化作用的结构方面。我们目前的工作集中在第I组内含子和代谢物响应性核酶GlmS。 GlmS核酶被小分子糖，葡糖胺-6-磷酸（GlcN 6P）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GlcN 6P产生的下调。GlmS核酶的RNA结构促进自我切割的精确化学机制尚不清楚。这种溶核核酶是否利用催化金属离子，例如I组内含子，或者它是否利用一个或多个杂环碱基用于一般酸或一般碱催化，例如HDV核酶？ GlcN 6P是否诱导构象变化以促进激活的RNA折叠，或者其功能基团之一，如氨基，直接参与化学反应？ 如果是后者，这将是第一个利用小分子辅因子促进化学反应的RNA的例子。 有趣的是，GlcN 6P的氨基是活性所绝对需要的，并且该胺的pKa随核酶的pKa而变化。 这种核酶的晶体结构将为这些生物化学问题提供重要的见解。 它还将为设计和解释该RNA的所有后续生物化学研究提供结构框架。

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.