TRNA RECOGNITION AND CATALYSIS BY E COLI HISRS

拟议工作的目标是了解阿尔斯如何利用 序列特异性RNA识别以获得最佳催化速率， 的特异性 最近的几份报告表明阿尔斯具有调节作用， 控制细胞生长和分裂的功能， 这可能与癌症问题有关。 在一份报告中，降低的HisRS或LysRS活性与G1 由于细胞周期蛋白D1表达减少而不是细胞周期蛋白E表达减少而导致的细胞停滞 表情 更清楚地了解这些酶如何在 分子水平可能最终导致癌症的发展 干预策略。 该项目的具体目标是：（1） 确定哪些氨基酸侧链与tRNA相互作用以及如何相互作用 单个突变影响氨酰化的总体速率 反应，2）通过以下步骤导出反应的自由能分布： 氨酰化反应各速率常数的测定 反应，和3）定量tRNA之间的偶联程度 识别和氨酰化，特别是在与 受体臂和A76受体核苷酸的取向。 这些 目标将通过使用前稳态动力学来实现 包括停流荧光测定法和猝灭流捕集法 实验

The objective of the proposed work is to understand how aaRS utilize sequence-specific RNA recognition for optimal catalytic rate and specificity. Several recent reports suggest that aaRS have regulatory functions in control over cell growth and division that were not previously noticed, and which may be relevant to the cancer problem. In one report, reduced HisRS or LysRS activity was associated with G1 arrest due to a reduction in cyclin D1 expression but not cyclin E expression. A clearer understanding of how these enzymes function at the molecular level may eventually lead to the development of cancer intervention strategies. The specific aims of the project are to 1) determine which amino acid side chains interact with the tRNA and how individual mutations affect the overall rate of the aminoacylation reaction, 2) derive a free energy profile for the reaction through determination of individual rate constants for the aminoacylation reaction, and 3) quantitate the extent of coupling between tRNA recognition and aminoacylation, particularly among contacts to the acceptor arm and the orientation of the A76 acceptor nucleotide. These aims will be carried out through the use of presteady state kinetics methods including stopped-flow fluorimetry and quenched-flow trapping experiments.