MECHANISM & MOLECULAR RECOGNITION OF ATRNA MODIFICATION

这项提议的目标是研究分子 TRNA-鸟氨酸转乙醇酶(Tgtase)反应的机理及 酶与核酸的分子相互作用 底物和小分子底物。这种酶参与了 TRNA的转录后修饰，并已与 分化和肿瘤发生。虽然这种酶已经被认为是 一段时间以来，还没有关于体外机制研究的报道。一个 更好地理解tRNA-鸟氨酸的作用机制 转甘醇酶可能提供对细胞外基质的生物学作用的洞察(S) TRNA的排队修饰。这些目标将通过以下方式实现 对大肠杆菌进行详细的体外动力学分析 Tgtase反应使用化学定义的、均匀的底物。 含有两种tRNA结构修饰的类似物 底物和小分子底物将被用来探测 Tgtase反应机理及分子决定因素研究 既有认知度又有反应性。一种体外转录系统 已经建立了使用T7RNA聚合酶来生产毫克 TRNA-Tyr(大肠杆菌)和结构类似物的数量。 小分子底物的结构-活性研究将被 对合成的底物和抑制剂类似物进行 与L.B.汤森德教授(密歇根大学， 药学院)。初步研究使用的是 Tgtase已在E. Coli.DNA扩增技术已被用于亚克隆tgt。 将基因转化为高效表达载体。这将方便生产。 足够数量的酶使更详细的研究 酶反应，并将允许生产突变酶用于 未来的研究。最终，这项研究将为 详细研究了酶之间的分子相互作用 和两种底物。这些调查将有助于澄清 蛋白质-核酸和蛋白质-Small的分子机制 分子识别和在酶催化中。

The objectives of this proposal are to investigate the molecular mechanism of the tRNA-guanine transglycolase (tgtase) reaction and the molecular interactions between the enzyme and both the nucleic acid substrate and the small molecule substrate. This enzyme is involved in posttranscriptional modification of tRNA and has been linked to differentiation and tumorigenesis. While this enzyme has been known for some time, no in vitro mechanistic investigations have been reported. A better understanding of the mechanism of action of tRNA-guanine transglycolase may provide insights into the biological role(s) of the queuine modification of tRNA. These objectives will be achieved by performing detailed, in vitro kinetic analyses of the Escherichia coli tgtase reaction using chemically-defined, homogeneous substrates. Analogues containing modifications of the structures of both the tRNA substrate and the small molecule substrate will be used to probe the mechanism of the tgtase reaction and to study the molecular determinants for both recognition and reactivity. An in vitro transcription system using the T7 RNA polymerase has been established to produce milligram quantities of tRNA-tyr (E. coli) and structural analogues. Structure-activity studies of the small molecule substrate will be performed on substrate and inhibitor analogues synthesized in collaboration with Professor L. B. Townsend (University of Michigan, College of Pharmacy). Preliminary studies have been performed using tgtase that has been isolated and purified as naturally expressed in E. coli. DNA amplification techniques have been used to subclone the tgt gene into an overexpressing vector. This will facilitate the production of sufficient quantities of enzyme enabling more detailed studies of the enzymic reaction and will allow for the production of mutant enzymes for future studies. Ultimately, this research will lay the groundwork for detailed investigations of the molecular interactions between the enzyme and both substrates. These investigations will help to elucidate the molecular mechanisms involved in protein-nucleic acid and protein-small molecule recognition and in enzymic catalysis.