Mechanistic Elucidation of RNA Modifying Enzymes

描述（由申请人提供）：所有生物体在转录后都会修饰其RNA，但特定修饰核苷的生理重要性及其产生机制仍不清楚。将进行两种这样的修饰核苷的产生的机制阐明：假尿苷（最常见的）和4-硫代尿苷（保护细菌免受紫外线照射）。假尿苷脱氢酶将RNA中的尿苷残基转化为假尿苷，即C-糖苷异构体。在所有生物体中都有许多这样的酶，它们分为六个家族。一种人假尿苷合酶dyskerin的突变或缺失导致先天性角化不良疾病，并且假尿苷残基在RNA中的广泛存在和保守证明了它们的生理重要性。新的调查扩大了我们的成功，对澄清的化学机制，其次是假尿苷酶和ThiI，负责4-硫代尿苷生成的酶。这些机制的知识本身作为化学先例是有价值的，因为它可以更有效地开发专门针对病原菌中这些酶的药物。特别是，最近的结果强烈反对的两个机制，占主导地位的思想如何假尿苷形成，同时提出了一个以前未认识到的可能性之一。将使用动力学同位素效应研究对这一新的替代方法进行测试。还将研究假尿苷合酶RluA对其各种底物的偏好，以探测允许该酶区分底物与非底物RNA的分子识别模式。一种新的机制，4-thiouridine代涉及二硫键连接的加合物之间的ThiI和RNA将进行测试，充分表征一个物种，在初步实验中匹配其属性。也将寻求新的机制重要的氨基酸残基的ThiI的身份。这些项目将由研究生和本科生研究人员进行，使他们接触到前沿的研究方法和思想。

DESCRIPTION (provided by applicant): All organisms modify their RNA after transcription, yet both the physiological importance of particular modified nucleosides and the mechanisms by which they are generated remain unknown. The mechanistic elucidation of the generation of two such modified nucleosides will be undertaken: pseudouridine (the most common) and 4-thiouridine (which protects bacteria against exposure to UV light). Pseudouridine synthases convert uridine residues in RNA into pseudouridine, the C-glycoside isomer. There are many such enzymes in all organisms, and they fall into six families. Mutation or deletion of one human pseudouridine synthase, dyskerin, leads to the disease dyskeratosis congenita, and the widespread occurrence and conservation of pseudouridine residues in RNA attests to their physiological importance. The new investigations extend our success towards the elucidation of the chemical mechanism followed by the pseudouridine synthases and ThiI, the enzyme responsible for 4-thiouridine generation. Knowledge of the mechanisms is valuable in its own right as a chemical precedent and because it may allow the more efficient development of pharmaceuticals that specifically targets these enzymes in pathogenic bacteria. In particular, recent results strongly disfavor one of the two mechanisms that had dominated thought on how pseudouridine is formed and simultaneously suggest a previously unrecognized possibility. This new alternative will be tested using kinetic isotope effect studies. The preference of the pseudouridine synthase RluA for its various substrates will also be investigated to probe the mode of molecular recognition that allows this enzyme to distinguish substrate from non-substrate RNA. A novel mechanism for 4-thiouridine generation involving a disulfide-linked adduct between ThiI and RNA will be tested by fully characterizing a species that match its properties in preliminary experiments. The identity of new mechanistically important amino acid residues of ThiI will also be sought. These projects will be undertaken by graduate students and by undergraduate researchers to expose them to cutting-edge research methods and thought.