Detection of anticodon nuclease residues involved in tRNALys cleavage specificity
Detection of anticodon nuclease residues involved in tRNALys cleavage specificity
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DOI：
10.1006/jmbi.1999.2634
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发表时间：
1999-04-02
影响因子：
5.6
通讯作者：
Kaufmann, G
中科院分区：
文献类型：
--
作者：
Meidler, R;Morad, I;Kaufmann, G
TRNA(Lys)特异性反密码子核酸酶以潜伏形式存在于含有可选的pur基因座的大肠杆菌菌株中。这种潜伏期是反密码子核酸酶核心多肽PrRC和IC型DNA限制性内切酶ECoprrI之间掩蔽相互作用的结果。T4噬菌体感染激活潜伏酶导致tRNA(Lys)5‘裂解到摆动碱基上，产生5’-OH和2‘，3’-环磷酸末端。PrRC的N-近端与(A/G)TPase和ECoprrI结合活性有关。因此，在C-Half处寻找与tRNA(Lys)识别和切割有关的残基。对编码200-313个PrRC残基的低G+C部分进行随机诱变，然后选择缺失依赖于反密码子核酸酶的致死性，并产生全长的PrRC样蛋白。这一过程产生了一系列错义突变，映射到PrRC和两个假定的脑膜炎奈瑟菌MC58同源物之间高度保守的区域。这一簇包括两个相邻的成员，它们放松了固有酶的切割特异性。我们还描述了另一种松弛的特异性模式，这是由于PRRC的过度表达。野生型prc和其他突变型等位基因也存在这种模式。在混杂条件下识别的额外底物通常具有类似tRNA(Lys)的反密码子。综上所述，这些数据表明，tRNA(Lys)的反密码子含有反密码子核酸酶识别元件，并暗示在它们的识别过程中PRRC中有一个保守的区域。(C)1999年学术出版社。
The tRNA(Lys)-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional pur locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNA(Lys) 5' to the wobble base, yielding 5'-OH and 2',3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNA(Lys) were searched for at the C-half. Random mutagenesis of the low-G + C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNA(Lys). Taken together, the data suggest that the anticodon of tRNA(Lys) harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition. (C) 1999 Academic Press.