Regulation of Substrate Binding and Catalysis in tRNase Z

描述（由申请人提供）：与其他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<$-内酰胺酶家族的成员，它去除3'端尾部，使得CCA可以通过tRNA核苷酸转移酶添加。3'端CCA是一种tRNase Z反决定簇，可确保成熟的tRNA顺利通过氨酰化。基序II中的残基是一个His簇（HxHxDH），是<$-内酰胺酶超家族的特征序列，是tRNase Z催化所需的，但不是底物前tRNA结合所需的。在基序II的羧基侧上的保守块中的残基（基序III、IV、V以及HEAT和HST环）涉及金属离子配位和催化，但不涉及底物结合。另一方面，基序II的氨基侧上的两个保守区块（PxKxRN环和基序I区域）参与底物结合和催化，并且可能有助于CCA抗决定。[Aim PxKxRN环和基序I区的先前研究将通过缺失PxKxRN环和通过组合两个区块中的突变来扩展。[Aim 2]由于tRNase Z的长型（tRNase ZL）被认为起源于短型（tRNase ZS）的串联基因复制，因此基序I区（？-）的改变形式被认为起源于短型（tRNase ZS）的串联基因复制。基序I）可以被发现朝向tRNase ZL的氨基末端。我们建议调查是否？-基序I参与tRNase Z催化。[Aim将通过蛋白酶质谱法研究野生型和变体tRNase Z，以分析环（包括PxKxRN环）与溶液中游离的酶和与tRNA结合的酶的暴露。[Aim人类线粒体tRNA的T环中天然发生的突变与母系传播的疾病和综合征，主要是肌病有关。将通过分析野生型和突变型线粒体底物的结构和tRNase Z加工来研究发病机制相关的T环置换的影响。实验程序，包括通过双延伸PCR的诱变、突变tRNase Z的杆状病毒表达和亲和纯化、体外转录和标记以制备前体tRNA底物、加工动力学的Michaelis-Menten分析、前体tRNA-tRNase Z复合物的Kd的凝胶位移测定、前体tRNA的结构探测、和蛋白酶-质谱分析，以评估在存在和不存在底物的情况下酶中环的暴露，将导致对生物医学上重要的酶反应的调节的详细了解。此外，这些方法将指导学生学员考虑在博士水平的生物医学研究事业。 转移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tRNase ZL（Elac 2，tRNA成熟途径中的酶之一）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 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 ¿-lactamase family of metal-dependent hydrolases, removes the 3' end trailer so that CCA can be added by tRNA nucleotidyltransferase. 3' end CCA, a tRNase Z anti-determinant, ensures that mature tRNA proceeds smoothly through aminoacylation. Residues in Motif II, a His cluster (HxHxDH) which is the signature sequence of the ¿-lactamase superfamily, are required for tRNase Z catalysis, but not for substrate pre-tRNA binding. Residues in conserved blocks on the carboxy side of Motif II (motifs III, IV, V and the HEAT and HST loops) are involved with metal ion coordination and catalysis but not with substrate binding. Two conserved blocks on the amino side of motif II (the PxKxRN loop and motif I region), on the other hand, are involved with both substrate binding and catalysis, and could contribute to CCA anti-determination. [Aim 1] Previous investigation of the PxKxRN loop and motif I region will be extended by deletion of the PxKxRN loop and by combining mutations in both blocks. [Aim 2] Because the long form of tRNase Z (tRNase ZL) is thought to have originated as a tandem gene duplication of the short form (tRNase ZS), an altered version of the motif I region (?-motif I) may be found toward the amino end of tRNase ZL. We propose to investigate whether ?-motif I participates in tRNase Z catalysis. [Aim 3] Wild type and variant tRNase Zs will be investigated by protease - mass spectroscopy to analyze the exposure of the loops, including the PxKxRN loop, with enzyme free in solution and bound to tRNA. [Aim 4] Naturally occurring mutations in the T loops of human mitochondrial tRNAs are associated with maternally transmitted diseases and syndromes, principally myopathies. Effects of pathogenesis-associated T loop substitutions will be investigated by analyzing structure and tRNase Z processing of wild type and mutant mitochondrial substrates. Experimental procedures, including mutagenesis by overlap-extension PCR, baculovirus expression and affinity purification of mutant tRNase Z, in vitro transcription and labeling to prepare pre-tRNA substrates, Michaelis-Menten analysis of processing kinetics, gel shift determination of Kd for pre-tRNA-tRNase Z complexes, structure probing of pre-tRNAs, and protease - mass spectroscopic analysis to evaluate exposure of loops in the enzyme in the presence and absence of substrate, will lead to detailed insight into the regulation of a biomedically significant enzyme reaction. Additionally, these methods will be instructive to student trainees considering biomedical research careers at the PhD level. PUBLIC HEALTH REVELANCE: 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 tRNase ZL (Elac2, one of the enzymes in the tRNA maturation pathway) has been associated with an elevated risk of prostate cancer, making the proposed research biomedically relevant.