Pseudouridine Synthases

描述(申请人提供)：假性尿苷是最常见的RNA转录后修饰，由假性尿苷合成酶催化尿苷异构化而产生。这些酶分为四个家族，它们没有全球序列相似性，但根据它们的三级结构似乎是同源的。一些假尿苷合成酶在原核生物和真核生物中是生理关键的，包括人类的dyskerin酶，它的缺失会导致X连锁疾病先天性角化不良。真核细胞中的假尿苷残基是真核生物中的一种重要功能，是转录前剪接所必需的。由于假尿苷合成酶家族的差异如此之大，它们仍然有可能通过不同的机制进行，这种差异最终可能被利用到新的抗生素类别中，或者提供对先天性角化不良疾病的更好理解。拟议的实验将阐明不同家族的两种大肠杆菌假尿苷合成酶RluA和TRUB所遵循的化学机制，这两种酶是机械工作的特别容易处理的靶标，因为它们处理小的RNA底物(茎环寡核苷酸)。含有5-氟尿苷的RNA已被用作假尿苷合成酶的抑制剂和机械探针，支持涉及Michael加成的机制。然而，最近的结果表明，RNA中的5-氟尿苷不能抑制TRUB，并且所提议的中间体不会发生假设的水解，这导致了对基于早期对同一化合物的研究的机理结论的怀疑。拟议的实验将解决关于RNA中5-氟尿苷的问题，并允许进一步的机械洞察。这种洞察力也将通过使用含有2‘-氟基的尿苷的RNA来获得，类似于使用氟糖来探索糖苷酶机制的一系列非常有意义的实验。结合核磁共振、质谱学、定点突变和动力学分析，辅以X射线结晶学，将研究这两种类型的氟化RNA以及似乎在催化中发挥作用的氨基酸残基的定点突变的效果。最后，将合成的小RNA底物用于检测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.