Residues in the conserved his domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding
Residues in the conserved his domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding
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DOI：
10.1016/j.jmb.2005.04.073
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发表时间：
2005-07-08
影响因子：
5.6
通讯作者：
Levinger, L
中科院分区：
文献类型：
--
作者：
Zareen, N;Yan, H;Levinger, L
转移rna作为前体转录，在5‘和3’端都有延伸。RNase P通过核内溶解去除5'端前导体。tRNase Z可以在tRNA成熟过程中通过核内溶解去除3′端尾链。CCA在真核生物、古细菌和一些细菌的trna中没有转录编码，必须在去除3'端尾链后通过添加CCA的酶添加。tRNase Z是金属依赖性水解酶p -内酰胺酶家族的一员，其特征序列保守的组氨酸簇（HxHxDH）对活性至关重要。从杆状病毒表达的果蝇tRNase Z开始，我们完成了His簇的18个残基Ala扫描，以分析这一关键区域的功能景观。根据取代对加工效率的影响，His簇内及其周围的残基可分为3类：8个残基的取代对加工效率影响不大，5个残基的取代对加工效率的影响中等（约为5-50倍），而5个保守残基（1个丝氨酸、3个组氨酸和1个天冬氨酸）的取代对加工效率的影响严重（约为500-5000倍）。使用凝胶位移测定的野生型和突变型解离常数（K-d值）没有显着差异，其数量级与km （220 nM）相同。在His域中替换所产生的较低的处理效率几乎完全是由于k（cat）值的降低；因此，tRNase Z的His簇中保守的、功能重要的残基参与了催化作用，而底物识别和结合功能必须位于蛋白质的其他位置。(c) 2005 Elsevier Ltd版权所有。
Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the P-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (similar to 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (similar to 500-5000-fold). Wild-type and mutant dissociation constants (K-d values), determined using gel shifts, displayed no substantial differences, and were of the same order as km (220 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced k(cat) values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein. (c) 2005 Elsevier Ltd. All rights reserved.