Regulation of Substrate Binding and Catalysis in Human tRNase Z

人 tRNase Z 底物结合和催化的调节

• 批准号: 7072040
• 负责人: LOUIS F LEVINGER
• 金额: $ 23.64万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072040
• 关键词: RNA; active sites; catalyst; crosslink; deafness; endonuclease; enzyme substrate; enzymes; family; gel; gene mutation; human; hydrolase; metals; mitochondria; mutant; nucleotides; proteins; transfer RNA

DESCRIPTION (provided by applicant): Like all other RNAs, transfer RNA is transcribed as a precursor and must undergo maturation. RNase P removes the 5' end leader. The endonuclease tRNase Z, a member of the (3-lactamase family of metal-dependent hydrolases, can remove the 3' end trailer. CCA can then be added by tRNA nucleotidyltransferase. CCA at the 3' end is a tRNase Z anti-determinant which ensures that mature tRNA proceeds smoothly through aminoacylation. Residues in Motif II, a His cluster (HxHxDH) which is the signature sequence of the (3-lactamase superfamily, are required for tRNase Z catalysis, but not for substrate pre-tRNA binding. Mutagenesis and recent crystal structures of tRNase Z suggest the involvement of residues in homology blocks upstream and downstream of Motif II in metal ion coordination, substrate binding and function of CCA in anti-determination/cleavage site selection. We propose to systematically scan 2 homology blocks of tRNase Z by substitution mutagenesis to establish the mechanism of CCA anti-determination. Additionally, naturally occurring mutations in the pre-tRNA 3' end trailer directly following the tRNase Z cleavage site in human mitochondrial tRNASer(UCN) are associated with maternally transmitted diseases such as non-syndromic deafness. These mutant sequences, like 3'-CCA, could interfere with a productive tRNase Z-substrate interaction. This hypothesis will be investigated by analyzing wild type and mutant substrates. The experimental procedures, including mutagenesis by overlap-extension PCR, baculovirus expression and affinity purification of mutant tRNase Z, Michaelis-Menten analysis of processing kinetics, gel shift determination of Kd for pre-tRNA-tRNase Z complexes, structure probing of pre-tRNAs, photo-crosslinking to identify specific contacts between enzyme and substrate and fluorescence methods to evaluate movement of both enzyme and substrate in the complex, will lead to detailed insight into the regulation of a biomedically significant enzyme reaction. Transfer RNA (tRNA) is central to the process of protein synthesis. Mutations in tRNAs are associated with maternally transmitted mitochondrial diseases and syndromes. Additionally, the gene that encodes 1 of the enzymes in the tRNA maturation pathway has been associated with an elevated risk of prostate cancer, making the proposed research biomedically relevant.

描述（由申请人提供）：与所有其他 RNA 一样，转移 RNA 作为前体进行转录，并且必须经历成熟。 RNase P 去除 5' 末端前导序列。核酸内切酶 tRNase Z 是金属依赖性水解酶 (3-内酰胺酶家族) 的成员，可以去除 3' 末端拖尾。然后可以通过 tRNA 核苷酸转移酶添加 CCA。3' 末端的 CCA 是 tRNase Z 抗决定簇这确保成熟的 tRNA 通过基序 II 中的残基（His 簇 (HxHxDH)）顺利进行，这是该基序的特征序列。 （3-内酰胺酶超家族，是 tRNase Z 催化所必需的，但不是底物前 tRNA 结合所必需的。tRNase Z 的诱变和最近的晶体结构表明，Motif II 上游和下游同源块中的残基参与金属离子配位、底物CCA 在抗决定/切割位点选择中的结合和功能我们建议通过取代诱变系统扫描 tRNase Z 的 2 个同源块，以建立 CCA 的机制。此外，紧随人类线粒体 tRNASer(UCN) 中 tRNA 酶 Z 切割位点的前 tRNA 3' 末端拖尾中自然发生的突变与非综合征性耳聋等母源性疾病相关。这些突变序列，如 3'-CCA，可能会干扰有效的 tRNase Z 底物相互作用。该假设将通过分析野生型和突变型底物进行研究。实验程序包括重叠延伸PCR诱变、杆状病毒表达和突​​变tRNase Z的亲和纯化、加工动力学的Michaelis-Menten分析、前tRNA-tRNase Z复合物Kd的凝胶位移测定、前tRNA的结构探测，光交联来识别酶和底物之间的特定接触，以及荧光方法来评估酶和底物在复合物中的运动，将导致对具有生物医学意义的酶的调节的详细了解 反应。转移 RNA (tRNA) 是蛋白质合成过程的核心。 tRNA 突变与母源性线粒体疾病和综合征有关。此外，编码 tRNA 成熟途径中一种酶的基因与前列腺癌风险升高相关，因此这项研究具有生物医学相关性。