Pseudouridine Synthases

描述（由申请人提供）：假尿苷是RNA最常见的转录后修饰，由假尿苷脱氢酶催化的尿苷异构化产生。这些酶分为四个家族，它们没有全局序列相似性，但在三级结构的基础上似乎是同源的。几种假尿苷激酶在原核生物和真核生物中是生理学上关键的，包括人酶dyskerin，其缺乏导致X连锁疾病先天性角化不良。小核RNA中的假尿苷残基是前mRNA剪接所必需的，这是真核生物中的一种基本功能。由于假尿苷激酶家族是如此高度分化，它们仍然有可能通过不同的机制进行，并且这种差异最终可能在新的抗生素类别中被利用，或者提供对先天性角化不良疾病的更好理解。本实验将阐明两个E.不同家族的大肠杆菌假尿苷酶，RluA和TruB，它们是机械工作的特别易处理的靶标，因为它们处理小RNA底物（茎环寡核苷酸）。含有5-氟尿苷的RNA已被用作假尿苷酶的抑制剂和机制探针，有利于涉及迈克尔加成的机制。然而，最近的研究结果表明，TruB是不抑制RNA中的5-氟尿苷，并假设水解的建议中间体不会发生，导致怀疑有关的机械结论的基础上，早期的研究与相同的化合物。所提出的实验将解决有关RNA中5-氟尿苷的问题，并允许进一步的机制洞察。使用含有具有2 '-氟基团的尿苷的RNA也将获得这种洞察力，类似于使用氟代糖来探测糖苷酶机制的一系列显著信息丰富的实验。结合核磁共振，质谱，定点诱变和动力学分析，辅以X-射线晶体学将被用来检查两种类型的氟化RNA的影响，以及定点诱变的氨基酸残基，似乎在催化中发挥作用。最后，合成的小RNA底物将用于检查RluA的底物特异性。

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.