Takaku Hiroaki, Minagawa Asako, Takagi Masamichi, Nashimoto Masayuki
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niitsu, Niigata 956-8603, Japan.
Nucleic Acids Res. 2004 Aug 18;32(15):4429-38. doi: 10.1093/nar/gkh774. Print 2004.
Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300-400 amino acids, and the other is a long form (tRNase ZL) that contains 800-900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA(Arg) and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the K(m) values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.
转运RNA（tRNA）3'加工内切核糖核酸酶（tRNase Z）是一种负责从pre-tRNA中去除3'拖尾序列的酶。tRNase Z存在两种类型：一种是由300 - 400个氨基酸组成的短形式（tRNase ZS），另一种是含有800 - 900个氨基酸的长形式（tRNase ZL）。在此，我们研究了短形式和长形式对各种RNA底物是否具有不同的偏好。我们检测了来自人类、大肠杆菌和海栖热袍菌的三种重组tRNase ZS，来自人类和酿酒酵母的两种重组tRNase ZL，来自猪肝的一种tRNase ZL，以及人类tRNase ZL的N端和C端半区，以检测它们对人类微小pre-tRNA（Arg）的切割作用以及RNase 65活性。所有tRNase ZL均能切割微小pre-tRNA并表现出RNase 65活性，而所有tRNase ZS以及人类tRNase ZL的两个半区除C端半区能勉强切割微小pre-tRNA外，均无法做到。我们还表明，只有tRNase Z的长形式能够在一种新型小向导RNA——钩状RNA的引导下特异性切割靶RNA。这些结果表明，tRNase ZL和tRNase ZS确实具有不同的底物特异性，且这些差异归因于tRNase ZL的N端半结构域。此外，tRNase ZS和tRNase ZL在NaCl、MgCl2和MnCl2的最佳浓度上存在差异，并且K(m)值表明tRNase ZL与pre-tRNA底物的相互作用比tRNase ZS更强。
