Regulation of Substrate Binding and Catalysis in tRNase Z

描述（由申请人提供）：像其他RNA一样，转移RNA作为前体转录，必须经过成熟。RNase P去除了5'端前导。内切酶tRNase Z是金属依赖性水解酶-内酰胺酶家族的一员，它去除3'端后链，以便通过tRNA核苷酸转移酶添加CCA。3'端CCA是一种tRNase Z的反决定因子，确保成熟的tRNA通过氨基酰化顺利进行。Motif II中的残基，一个His簇（HxHxDH）是内酰胺酶超家族的特征序列，是tRNase Z催化所必需的，但不是底物前trna结合所必需的。Motif II羧基侧保守区（Motif III、IV、V以及HEAT和HST环）的残基与金属离子配位和催化有关，但与底物结合无关。另一方面，基序II氨基侧的两个保守区域（PxKxRN环和基序I区域）既参与底物结合又参与催化作用，可能有助于CCA抗测定。[目的1]先前对PxKxRN环和motif I区域的研究将通过删除PxKxRN环和结合两个区域的突变来扩展。[目的2]由于tRNase Z的长链（tRNase ZL）被认为起源于短链（tRNase ZS）的串联基因复制，基序I区域的改变版本(？-motif I)可能在tRNase ZL的氨基端发现。我们建议调查是否？-motif I参与tRNase Z催化。[目的3]将通过蛋白酶-质谱法研究野生型和变体tRNase Zs，以分析包括PxKxRN环在内的环在溶液中游离酶并与tRNA结合的暴露情况。[目的4]人类线粒体trna的T环中自然发生的突变与母体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加工来研究致病相关T环替换的影响。实验程序，包括重叠延伸PCR诱变、杆状病毒表达和突变体tRNase Z的亲和纯化、制备前trna底物的体外转录和标记、加工动力学的Michaelis-Menten分析、前trna -tRNase Z复合物的Kd的凝胶移位测定、前trna的结构探测、蛋白酶-质谱分析以评估在底物存在和缺失的情况下酶环的暴露。将导致详细了解生物医学上重要的酶反应的调节。此外，这些方法将对考虑在博士阶段从事生物医学研究的学生有指导意义。

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.