Mechanistic Elucidation of RNA Modifying Enzymes

描述(申请人提供)：所有生物在转录后都会修改它们的RNA，但特定修饰的核苷的生理重要性和产生它们的机制仍然未知。将从机理上阐明两种这样的修饰核苷的产生：假尿苷(最常见)和4-硫代尿苷(保护细菌免受紫外线照射)。假尿苷合成酶将RNA中的尿苷残基转化为假尿苷，即C-糖苷异构体。所有生物体中都有许多这样的酶，它们分为六个家族。人类假性尿苷合成酶Dyskerin的突变或缺失导致先天性角化不良，而RNA中假性尿苷残基的广泛存在和保守证明了它们的生理意义。这些新的研究将我们的成功扩展到阐明了假性尿苷合成酶和ThiI的化学机制，ThiI是负责产生4-硫代尿苷的酶。对这些机制的了解本身就是一个有价值的化学先例，因为它可能允许更有效地开发专门针对病原细菌中的这些酶的药物。特别是，最近的结果强烈反对两种机制中的一种，这两种机制曾主导着关于假尿苷如何形成的想法，同时也暗示了一种以前未被认识到的可能性。这一新的替代方案将通过动力学同位素效应研究进行测试。假尿苷合成酶RluA对其各种底物的选择性也将被研究，以探索使该酶区分底物和非底物RNA的分子识别模式。一种新的生成4-硫代尿苷的机制，涉及ThiI和RNA之间的二硫键加合物，将通过在初步实验中完全表征与其性质匹配的物种来进行测试。还将寻找ThiI的新的机械上重要的氨基酸残基的身份。这些项目将由研究生和本科生研究人员承担，让他们接触到尖端的研究方法和思想。

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.