Dutta Tanmay, Deutscher Murray P
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33101, USA.
J Biol Chem. 2009 Jun 5;284(23):15425-31. doi: 10.1074/jbc.M109.005462. Epub 2009 Apr 14.
Processing of the 3' terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3'-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3'-CCA sequence or a 3'-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3' fragments as short as 4 nucleotides. Although the presence of a 3'-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.
许多生物体中tRNA 3'末端的加工是由一种称为RNase Z或3'-tRNase的核糖核酸内切酶完成的，该酶在鉴别核苷酸后进行切割，以便添加通用的-CCA序列。在一些真细菌中，如大肠杆菌，所有已知的tRNA基因中都编码了-CCA序列。然而，即使tRNA成熟不需要其作用，仍然存在一种RNase Z同源物（RNase BN）。为了帮助确定哪些RNA分子可能是RNase BN的潜在底物，我们使用各种合成底物对其特异性和催化潜力进行了详细研究。我们在此表明，RNase BN对双链和单链RNA均有活性，但双链RNA是其偏好的底物。该酶表现出很强的碱基特异性，对连续的C残基没有活性。3'-CCA序列或3'-磷酸基团的存在会强烈抑制RNase BN。RNase BN消化产生的极限产物是三聚体，但对于长度小于10个核苷酸的分子，消化速率会减慢。最有趣的是，RNase BN在某些底物上作为一种分布性外切核糖核酸酶起作用，释放单核苷酸和一系列消化产物。然而，RNase BN也能进行内切切割，释放短至4个核苷酸的3'片段。虽然3'-磷酸基团的存在消除了外切核糖核酸酶的作用，但对内切核糖核酸酶切割没有影响。这些数据表明RNase BN可能与RNase Z家族的其他成员不同，它们为确定该酶的生理作用提供了重要信息以供参考。
