MECHANISM & MOLECULAR RECOGNITION OF ATRNA MODIFICATION

本提案的目的是研究分子 tRNA-guanine transglycolase（tgtase）反应的机理和 酶和核酸之间的分子相互作用 基质和小分子基质。 这种酶参与了 转录后修饰的tRNA，并已连接到 分化和肿瘤发生。 虽然这种酶已经被称为 一段时间以来，没有报道体外机理研究。 一 更好地理解tRNA-鸟嘌呤的作用机制 糖基转移酶的研究可以提供对糖基转移酶的生物学作用的深入了解。 tRNA的精氨酸修饰。 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 使用化学上确定的均质底物的TGtase反应。 含有两种tRNA结构修饰的类似物 底物和小分子底物将用于探测 TGtase反应的机理和研究分子决定簇 用于识别和反应。 体外转录系统 已经建立了使用T7 RNA聚合酶来产生毫克 tRNA-tyr（E. coli）和结构类似物。 小分子底物的结构-活性研究将在 对底物和抑制剂类似物进行合成， 与L教授合作。B。汤森（密歇根大学， 药学院）。 初步研究已经使用 已分离和纯化的天然表达于E. 杆菌 DNA扩增技术已被用于亚克隆tgt 基因导入过表达载体。 这将有助于生产 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， 未来的研究。 最终，这项研究将奠定基础， 详细研究了这种酶与 和两个衬底。 这些调查将有助于阐明 蛋白质-核酸和蛋白质-小分子的分子机制 分子识别和酶催化。

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.