Pseudouridine Synthases

描述（由申请人提供）：伪尿嘧啶是RNA最常见的转录后修饰，由伪尿嘧啶合成酶催化尿嘧啶异构化产生。这些酶分为四个家族，它们没有全局序列相似性，但根据它们的三级结构看起来是同源的。几种假尿嘧啶合成酶在原核生物和真核生物中具有重要的生理作用，包括人类dyskerin酶，缺乏这种酶会导致先天性角化不良症。小核RNA中的假尿嘧啶残基是前mrna剪接所必需的，这是真核生物的基本功能。由于假尿嘧啶合成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，这种差异可能最终被利用在新的抗生素类别中或提供对先天性角化不良病的更好理解。这些实验将阐明两种不同家族的大肠杆菌假尿嘧啶合成酶（RluA和TruB）的化学机制，这两种合成酶处理小RNA底物（茎环寡核苷酸），是机制工作中特别容易处理的靶点。含有5-氟吡啶的RNA已被用作假吡啶合成酶的抑制剂和机制探针，倾向于涉及迈克尔加成的机制。然而，最近的结果表明，TruB在RNA中不受5-氟吡啶的抑制，并且假设的中间体没有发生水解，这导致人们对基于对同一化合物的早期研究得出的机制结论产生怀疑。提出的实验将解决有关RNA中5-氟吡啶的问题，并允许进一步了解机制。使用含有2'-氟基团尿苷的RNA也可以获得这样的见解，类似于使用氟糖来探测糖苷酶机制的一系列非常有用的实验。将采用核磁共振、质谱、定点诱变和辅以x射线晶体学的动力学分析相结合的方法来检查两种氟化RNA的效果以及似乎在催化中起作用的氨基酸残基的定点诱变。最后，合成的小RNA底物将用于检测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.