Regulation of Substrate Binding and Catalysis in Human tRNase Z

描述（由申请人提供）：与所有其他RNA一样，转运RNA作为前体转录，必须经历成熟。RNase P去除5'末端前导序列。内切核酸酶tRNase Z是金属依赖性水解酶的β-内酰胺酶家族的成员，可以去除3'端尾部。然后可以通过tRNA核苷酸转移酶添加CCA。3'端的CCA是一个tRNase Z反决定簇，它确保成熟的tRNA通过氨酰化顺利进行。基序II中的残基是β-内酰胺酶超家族的特征序列His簇（HxHxDH），其是tRNase Z催化所需的，但不是底物前tRNA结合所需的。tRNase Z的突变和最近的晶体结构表明，在金属离子配位，底物结合和CCA的功能，在抗决定/切割位点选择的模体II的上游和下游的同源块中的残基的参与。我们建议通过置换突变系统扫描tRNase Z的2个同源区段，以建立CCA抗决定的机制。此外，直接在人线粒体tRNASer（UCN）中tRNase Z切割位点之后的pre-tRNA 3'末端尾部中天然存在的突变与母体传播的疾病如非综合征性耳聋相关。这些突变序列，如3 '-CCA，可以干扰生产性tRNase Z-底物相互作用。将通过分析野生型和突变体底物来研究这一假设。本文介绍了用双延伸PCR诱变、杆状病毒表达和亲和纯化突变型tRNase Z、Michaelis-Menten动力学分析、凝胶位移测定、结构分析、光交联法鉴定酶与底物的特异性接触、荧光法测定酶与底物在复合物中的运动等实验方法。将导致对生物医学上重要的酶反应的调节的详细了解。转运RNA（tRNA）是蛋白质合成过程的核心。tRNA的突变与母系传播的线粒体疾病和综合征有关。此外，编码tRNA成熟途径中的一种酶的基因与前列腺癌的风险升高有关，使拟议的研究具有生物医学相关性。

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.