Steiger M A, Kierzek R, Turner D H, Phizicky E M
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
Biochemistry. 2001 Nov 20;40(46):14098-105. doi: 10.1021/bi011388t.
The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.
酿酒酵母中tRNA剪接的最后一步需要2'-磷酸转移酶（Tpt1）将连接的tRNA上的2'-磷酸转移至NAD，生成成熟的tRNA和ADP核糖-1''-2''-环磷酸酯。为了探究Tpt1蛋白如何识别底物RNA，我们测定了Tpt1蛋白与2'-磷酸化连接tRNA及多种相关底物的稳态动力学参数。Tpt1蛋白对连接的tRNA具有较高的表观亲和力（K(m,RNA)，0.35 nM）和较低的周转速率（k(cat)，0.3 min(-1)）。Tpt1蛋白既能识别tRNA，也能识别RNA内部的2'-磷酸。稳态动力学分析表明，随着RNA结构和长度的丧失，K(m,RNA)和k(cat)均相应增加。对于源自连接tRNA反密码子茎环的2'-磷酸化十八聚体，K(m,RNA)和k(cat)分别比连接的tRNA高5倍和8倍，而对于像pApA(p)pA这样的简单底物，K(m,RNA)和k(cat)分别高430倍和150倍。Tpt1对具有末端5'-或3'-磷酸的三聚体无明显活性，并且除非存在相邻的3'-磷酸或磷酸二酯，否则去除末端2'-磷酸的效率非常低。Tpt1的K(m,NAD)取决于底物：连接tRNA时K(m,NAD)为10 μM，pApA(p)pA时为200 μM，pApApA(p)时为600 μM。对来自大肠杆菌的功能性Tpt1蛋白同源物KptA的初步分析表明，尽管未知大肠杆菌具有2'-磷酸化RNA底物，但KptA蛋白在动力学参数上与酵母Tpt1极为相似。
