TRNA RECOGNITION AND CATALYSIS BY E COLI HISRS

大肠杆菌 HISRS 的 TRNA 识别和催化

• 批准号: 6178859
• 负责人: MICHAEL L BOVEE
• 金额: $ 3.92万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-16 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6178859
• 关键词: Escherichia coli; aminoacid tRNA ligase; chemical kinetics; enzyme activity; histidine; polymerase chain reaction; site directed mutagenesis; stop flow technique; transfer RNA

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.

拟议工作的目标是了解AARS如何利用 特定于序列的RNA识别，可实现最佳催化速率和 专一性。最近的几份报告表明，Aars有监管机构 控制细胞生长和分裂的功能 之前注意到的，这可能与癌症问题有关。 在一份报告中，HisRS或LysRS活性降低与G1相关 细胞周期蛋白D1表达减少而非细胞周期蛋白E表达受阻 表情。更清楚地了解这些酶是如何在 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 个别突变会影响氨基酰化的总体速度。 反应，2)导出反应的自由能分布，通过 氨基酰化反应个别速率常数的测定 反应，以及3)定量tRNA之间的偶联程度 识别和氨基酰化，特别是在与 受体臂和A76受体核苷酸的定位。这些 AIMS将通过使用稳态前动力学来实现 停流荧光法和猝灭流动捕集法 实验。