Genschik P, Drabikowski K, Filipowicz W
Friedrich Miescher-Institut, P. O. Box 2543, 4002 Basel, Switzerland.
J Biol Chem. 1998 Sep 25;273(39):25516-26. doi: 10.1074/jbc.273.39.25516.
The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of various RNA substrates. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity (Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W. (1997) EMBO J. 16, 2955-2967). Analysis of the requirements and substrate specificity of the E. coli protein, presented in this work, demonstrates that properties of the bacterial and human enzymes are similar. ATP is the best cofactor (Km = 20 microM), whereas GTP (Km = 100 microM) and other nucleoside triphosphates (NTPs) act less efficiently. The enzyme undergoes nucleotidylation in the presence of [alpha-32P]ATP and, to a lesser extent, also in the presence of other NTPs. Comparison of 3'-phosphorylated oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are at least 300-fold poorer substrates for the enzyme. The E. coli cyclase gene, named rtcA, forms part of an uncharacterized operon containing two additional open reading frames (ORFs). The ORF positioned immediately upstream, named rtcB, encodes a protein that is also highly conserved between Eucarya, Bacteria, and Archaea. Another ORF, called rtcR, is positioned upstream of the rtcA/rtcB unit and is transcribed in the opposite direction. It encodes a protein having features of sigma54-dependent regulators. By overexpressing the N-terminally truncated form of RtcR, we demonstrate that this regulator indeed controls expression of rtcA and rtcB in a sigma54-dependent manner. Also consistent with the involvement of sigma54, the region upstream of the transcription start site of the rtcA/rtcB mRNA contains the -12 and -24 elements, TTGCA and TGGCA, respectively, characteristic of sigma54-dependent promoters. The cyclase gene is nonessential as demonstrated by knockout experiments. Possible functions of the cyclase in RNA metabolism are discussed.
RNA 3'-末端磷酸环化酶催化各种RNA底物末端的3'-磷酸依赖ATP转化为2',3'-环磷酸二酯。最近克隆的编码人环化酶的cDNA表明，编码环化酶样蛋白的基因在真核生物、细菌和古细菌中是保守的。大肠杆菌基因编码的蛋白质被过度表达，并显示具有RNA 3'-磷酸环化酶活性（Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W. (1997) EMBO J. 16, 2955 - 2967）。本文对大肠杆菌蛋白质的需求和底物特异性进行了分析，结果表明细菌和人源酶的性质相似。ATP是最佳辅因子（Km = 20 microM），而GTP（Km = 100 microM）和其他核苷三磷酸（NTPs）的作用效率较低。该酶在[α-32P]ATP存在下会发生核苷酸化，在其他NTPs存在下程度较小。对相同序列的3'-磷酸化寡核糖核苷酸和寡脱氧核糖核苷酸进行比较表明，后者作为该酶的底物至少差300倍。大肠杆菌的环化酶基因名为rtcA，是一个未鉴定操纵子的一部分，该操纵子还包含另外两个开放阅读框（ORFs）。紧邻上游的ORF名为rtcB，编码一种在真核生物、细菌和古细菌中也高度保守的蛋白质。另一个名为rtcR的ORF位于rtcA/rtcB单元的上游，转录方向相反。它编码一种具有sigma54依赖性调节因子特征的蛋白质。通过过度表达RtcR的N端截短形式，我们证明该调节因子确实以sigma54依赖性方式控制rtcA和rtcB的表达。同样与sigma54的参与一致，rtcA/rtcB mRNA转录起始位点上游区域分别包含-12和-24元件，即TTGCA和TGGCA，这是sigma54依赖性启动子的特征。敲除实验表明环化酶基因是非必需的。文中讨论了环化酶在RNA代谢中的可能功能。
