STRUCTURAL EFFECTS OF MODIFIED NUCLEOTIDES ON THE ACTIVE SITE/FOLD OF THE HAIRPI

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。 发夹状核酶是一种小的催化RNA基序，它不直接利用金属离子进行断键。这表明作用机制涉及：（i）过渡态稳定，（ii）一般酸/碱催化或（iii）特定酸/碱催化，可能通过活化水。 申请人的实验室已经在几种核苷酸变体的存在下化学合成并结晶了最小发夹核酶。A38 C、A38 isoG、A38 AP（即氨基嘌呤）导致功能丧失，而A38 DAP（di-AP）在活性测定中导致功能获得。 这些变体的晶体已经以呈现<$pre-catalytic <$and <$transition-state <$geometry的形式制备。先前，最小发夹核酶结构由申请人的实验室以2 ½分辨率确定。 从这一建议产生的衍射数据将确定结构特征（如有序沃茨和相互作用距离），这些特征被用来支持一种作用机制。

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 hairpin ribozyme is small catalytic RNA motif that does not utilize metal ions directly in bond scission. This suggests a mechanism of action involving: (i) transition-state stabilization, (ii) general acid/base catalysis or (iii) specific acid/base catalysis, possibly through activated water(s). The applicant¿s lab has chemically synthesized and crystallized a minimal hairpin ribozyme in the presence of several nucleotide-variants. A38C, A38isoG, A38AP (i.e. aminopurine) resulted in loss-of-function, whereas A38DAP (di-AP) resulted in a gain-of-function in activity assays. Crystals of these variants have been prepared in forms presenting the ¿pre-catalytic¿ and ¿transition-state¿ geometries. Previously, the minimal hairpin ribozyme structure was determined at 2 ¿ resolution by the applicant's lab. The diffraction data resulting from this proposal will identify structural features (such as ordered waters and interaction distances) that have been invoked to support one mechanism of action over another.