tRNA（尿嘧啶-5-）-甲基转移酶与NO2Ura-tRNA的相互作用。
Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA.
作者信息
Gu X, Matsuda A, Ivanetich K M, Santi D V
机构信息
Department of Biochemistry, University of California, San Francisco 94143-0448 USA.
出版信息
Nucleic Acids Res. 1996 Mar 15;24(6):1059-64. doi: 10.1093/nar/24.6.1059.
tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an in vitro transcription reaction. The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic mechanism. When assayed shortly after mixing, the tRNA analog, NO2Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT). Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate. Covalent RUMT-NO2Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE. The interaction of RUMT and NO2Ura-tRNA was deduced to involve formation of a reversible complex, followed by formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO2Ura 54 in NO2Ura-tRNA.
通过在体外转录反应中用5-硝基三磷酸尿苷（5-nitro-UTP）替代三磷酸尿苷（UTP），制备了尿嘧啶完全被5-硝基尿嘧啶取代的转运核糖核酸（tRNA）。其基本原理是5-硝基取代基使尿嘧啶杂环的6-碳对亲核试剂具有活性，因此可以提供基于机制的酶抑制剂，这些酶在其催化机制中利用了这一特性。在混合后不久进行检测时，tRNA类似物NO2Ura-tRNA是tRNA-尿嘧啶甲基转移酶（RUMT）的有效竞争性抑制剂。孵育后，该类似物会导致RUMT的时间依赖性失活，这种失活可通过稀释到大量过量的tRNA底物中而逆转。共价RUMT-NO2Ura-tRNA复合物可以在硝酸纤维素滤膜上或通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离。推断RUMT与NO2Ura-tRNA的相互作用涉及形成可逆复合物，随后形成可逆共价复合物，其中RUMT的半胱氨酸324与NO2Ura-tRNA中的NO2Ura 54的6-位相连。
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