Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3' end processing.
Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3' end processing.
复制标题
DOI：
10.1261/rna.4206
复制
发表时间：
2006-06
期刊：
影响因子：
4.5
通讯作者：
N. Zareen;A. Hopkinson;L. Levinger
中科院分区：
文献类型：
--
作者：
N. Zareen;A. Hopkinson;L. Levinger
TRNase Z具有金属依赖的β-内酰胺酶家族的His结构域(HxHxDh；Motif II)，它可以内切去除前tRNA3‘端拖尾。基序II与基序III-V在其羧基一侧结合，配位两个二价金属离子，构成催化核心。已有研究表明，Motif II氨侧的PxKxRN环和Motif I可以调节tRNase Z的活性，包括CCA在成熟tRNA中的反决定簇作用。ALA遍历这两个同源区块，揭示了其中的取代位意外降低了催化效率的残基。虽然基序II的替换可以显著影响k(CAT)而不影响k(M)，但随着PxKxRN环和基序I中几个保守残基的替换，k(M)增加了5到15倍。这些k(M)的增加表明了底物结合的模型。表达的tRNase Z处理3‘端带有CCA的成熟tRNA的效率比具有3’端自然序列的前tRNA低约80倍，这是因为减少了k(CAT)，而对k(M)没有影响，表明CCA反决定簇是该酶的特征。
tRNase Z, which can endonucleolytically remove pre-tRNA 3'-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the beta-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k(cat) without affecting k(M), five- to 15-fold increases in k(M) are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k(M) suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3' end approximately 80 times less efficiently than a pre-tRNA possessing natural sequence of the 3'-end trailer, due to reduced k(cat) with no effect on k(M), showing the CCA anti-determinant to be a characteristic of this enzyme.