What is the function of distinct nucleobases in the catalytic mechanism of the hairpin ribozyme?

发夹核酶催化机制中不同核碱基的功能是什么？

• 批准号: 209933141
• 负责人: Professorin Dr. Stephanie Kath-Schorr
• 金额: --
• 依托单位: Institut für Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/209933141?language=en
• 关键词: What; function; distinct; nucleobases; catalytic

RNA catalysis plays an important role in processing and translation of ribonucleic acids but the catalytic mechanism remains unclear in most cases. The hairpin ribozyme catalyzes cleavage of RNA releasing a fragment with a free 5-OH group and one with a 2-3-cyclic phosphate. This research proposal addresses the question if the catalytic mechanism of the hairpin ribozyme is based on general acid-base catalysis by the distinct nucleobases A38 (acid) and G8 (base) or if these purine bases only provide electrostatic stabilization of the transition state. Different approaches will be used to address this question. In all cases, hairpin ribozymes will be assembled from chemically synthesized RNA molecules. First of all, a substrate will be synthesized in which the 5-oxygen of the scissile phosphate is replaced by sulfur (5-PS). This highly activated substrate will not require general acid catalysis for its cleavage and replacement of the acid A38 by other nucleobases should not inhibit the catalytic function. Secondly, the 5-PS substrate will allow to investigate the pH profile of the reaction independently from the acid. Then, ribozymes with modified nucleobases of lower pKa at the G8 position could be used to proof the role of G8 as general base. A third approach will be to concentrate on the transition state of the reaction. For this, kinetic isotope effects of reacting atoms will be investigated. In summary, using a combination of chemistry and the methods of mechanistic enzymology, it should be possible to elucidate the catalytic mechanism of the hairpin ribozyme. Understanding the catalytic mechanisms of different ribozymes is important for several reasons. First, they perform reactions necessary for the metabolism in living cells. Second, they can potentially provide a glimpse into the evolution of life and the possible existence of an RNA world. Lastly, as simplified macromolecular catalysts they provide mechanistic insight into biocatalysis in general.

RNA催化在核糖核酸的加工和翻译过程中起着重要的作用，但其催化机制在大多数情况下仍不清楚。发夹核酶催化RNA的切割，释放具有游离5-OH基团的片段和具有2-3-环磷酸的片段。该研究方案解决了发夹状核酶的催化机制是否基于不同核碱基A38（酸）和G8（碱）的一般酸碱催化的问题，或者这些嘌呤碱基是否仅提供过渡态的静电稳定。将采用不同的方法来解决这一问题。在所有情况下，发夹核酶将从化学合成的RNA分子组装。首先，将合成底物，其中易断裂的磷酸盐的5-氧被硫（5-PS）取代。这种高度活化的底物不需要一般的酸催化来裂解，并且用其它核碱基取代酸A38不应抑制催化功能。其次，5-PS底物将允许独立于酸研究反应的pH曲线。然后，在G8位置具有较低pKa的修饰的核碱基的核酶可以用于证明G8作为通用碱基的作用。第三种方法是集中研究反应的过渡态。为此，将研究反应原子的动力学同位素效应。总之，结合化学和机械酶学的方法，有可能阐明发夹状核酶的催化机制。了解不同核酶的催化机制有几个重要的原因。首先，它们在活细胞中进行新陈代谢所必需的反应。第二，它们可以提供对生命进化和RNA世界可能存在的一瞥。最后，作为简化的大分子催化剂，它们提供了一般生物催化的机理见解。