Pseudouridine Synthases

假尿苷合成酶

• 批准号: 7157611
• 负责人: EUGENE G MUELLER
• 金额: $ 9.7万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7157611
• 关键词: 5 fluorouridine; Address; Amino Acids; Antibiotics; Anticodon; Behavior; Binding; Catalysis; Chemicals; Class; Crystallography; Disease; Dyskeratosis Congenita; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Event; Family; Fluorine; Glycoside Hydrolases; Heterogeneous Nuclear RNA; Human; Hydrolysis; Investigation; Kinetics; Link; Mass Spectrum Analysis; Methods; Oligonucleotides; Physiological; Play; Positioning Attribute; Post-Transcriptional RNA Processing; Prokaryotic Cells; Pseudouridine; Pyrimidine; Pyrimidines; RNA; RNA Splicing; RNA-Protein Interaction; Reaction; Research Personnel; Ribose; Role; Series; Side; Site-Directed Mutagenesis; Small Nuclear RNA; Small RNA; Specificity; Structure; Substrate Specificity; Testing; Transfer RNA; Uncertainty; Uridine; Work; X-Ray Crystallography; base; chemical kinetics; falls; inhibitor/antagonist; insight; mRNA Precursor; programs; research study; stem

DESCRIPTION (provided by applicant): Pseudouridine is the most common post-transcriptional modification of RNA, and arises from the isomerization of uridine catalyzed by the pseudouridine synthases. These enzymes fall into four families that share no global sequence similarity but appear homologous on the basis of their tertiary structures. Several pseudouridine synthases are physiologically critical in prokaryotes and eukaryotes, including the human enzyme dyskerin, the absence of which causes the X-linked disease dyskeratosis congenita. Pseudouridine residues in small nuclear RNA are required for pre-mRNA splicing, an essential function in eukaryotes. Since the families of pseudouridine synthases are so highly divergent, it remains possible that they proceed by different mechanisms, and such differences may eventually be exploited in new classes of antibiotics or provide a better understanding of the disease dyskeratosis congenita. The proposed experiments will elucidate the chemical mechanism followed by two E. coli pseudouridine synthases of different families, RluA and TruB, which are particularly tractable targets for mechanistic work because they handle small RNA substrates (stem-loop oligonucleotides). RNA containing 5-fluorouridine has been used as an inhibitor and mechanistic probe of the pseudouridine synthases, favoring a mechanism involving a Michael addition. Recent results, however, indicate that TruB is not inhibited by 5-fluorouridine in RNA and that an assumed hydrolysis of a proposed intermediate does not occur, leading to doubt concerning the mechanistic conclusions based on earlier studies with the same compound. The proposed experiments will resolve issue concerning 5-fluorouridine in RNA and allow further mechanistic insight. Such insight will also be gained using RNA containing uridine with a 2'-fluoro group, similar to the series of remarkably informative experiments using fluorosugars to probe the mechanism of glycosidases. A combination of NMR, mass spectrometry, site-directed mutagenesis and kinetic analysis supplemented by X-ray crystallography will be employed to examine the effects of the two types of fluorinated RNA as well as site-directed mutagenesis of amino acid residues that appear to play a role in catalysis. Finally, synthesized small RNA substrates will be used to examine the substrate specificity of RluA.

描述（由申请人提供）：假氨酸是RNA的最常见的转录后修饰，并且是由尿苷的异构化引起的，尿苷的异构化是由假硫氨酸合成酶催化的。这些酶落入四个没有具有全球序列相似性的家族，但根据其三级结构而显得同源。在原核生物和真核生物中，包括人类酶异性克林在生理上至关重要，在生理上至关重要。小核RNA中的假喹啉残基是MRNA剪接需要的，这是真核生物中的重要功能。由于伪氨酸合酶的家族非常不同，因此仍然有可能通过不同的机制进行，并且最终可能在新的抗生素类别中利用这种差异，或者对疾病疾病的疾病康涅狄格病。提出的实验将阐明化学机制，然后是不同家族的两个大肠杆菌假氨酸合成酶，RLUA和TRUB，这是机械工作特别可触及的靶标，因为它们处理了小的RNA底物（STEM-LOOP寡核苷酸）。含有5-氟苷的RNA已被用作假氨酸合酶的抑制剂和机械探针，有利于一种涉及迈克尔添加的机制。然而，最近的结果表明，trub在RNA中没有抑制5-氟嘧啶，并且不会发生假定的对拟议中间体的水解，从而对基于早期的相同化合物研究的机械结论产生了疑问。提出的实验将解决有关RNA中5-氟岛的问题，并允许进一步的机械洞察力。这种洞察力还将使用含有2'-氟体组的尿苷的RNA获得，类似于使用氟胡萝卜素进行一系列信息丰富的实验，以探测糖苷酶的机制。将使用NMR，质谱，定位诱变和补充X射线晶体学的动力学分析的组合来检查两种类型的氟RNA以及氨基酸残基的位置指导诱变的作用，这些诱变似乎在催化中起着似乎在催化中起作用的作用。最后，合成的小RNA底物将用于检查RLUA的底物特异性。