Characterization of the Escherichia coli RNA 3′-terminal phosphate cyclase and its σ54-regulated operon
Characterization of the Escherichia coli RNA 3′-terminal phosphate cyclase and its σ54-regulated operon
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DOI：
10.1074/jbc.273.39.25516
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发表时间：
1998-09-25
影响因子：
4.8
通讯作者：
Filipowicz, W
中科院分区：
文献类型：
--
作者：
Genschik, P;Drabikowski, K;Filipowicz, W
RNA 3'-末端磷酸环化酶催化各种 RNA 底物末端的 3'-磷酸盐依赖 ATP 转化为 2',3'-环状磷酸二酯。最近克隆编码人环化酶的 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被过表达并显示出具有 RNA 3'-磷酸环化酶活性（Genschik, P,, Billy, E,, Swianiewicz, M, 和 Filipowicz, W, (1997) EMBO J. 16, 2955-2967），对大肠杆菌蛋白的要求和底物特异性的分析，在这项工作中提出，表明细菌和人类酶的性质相似，ATP 是最好的辅助因子（K-m = 20） mu M)，而 GTP (K-m = 100 mu M) 和其他三磷酸核苷 (NTP) 的作用效率较低，该酶在 [α-P-32]ATP 存在下发生核苷酸化，并且在较小程度上也在其他 NTP 存在下发生核苷酸化。 3'-磷酸化寡核糖核苷酸和相同序列的寡脱氧核糖核苷酸的比较表明，后者对于酶的底物至少差300倍。大肠杆菌环化酶基因（名为 rtcA）构成未表征操纵子的一部分，其中包含两个额外的开放阅读框（ORF）。位于紧邻上游的 ORF（名为 rtcB）编码一种在真核生物、细菌和古细菌之间也高度保守的蛋白质。另一个 ORF，称为 rtcR，位于 rtcA/rtcB 单元的上游，并以相反方向转录。它编码具有 sigma(54) 依赖性调节因子特征的蛋白质。通过过表达 RtcR 的 N 端截短形式，我们证明该调节因子确实以 sigma(54) 依赖性方式控制 rtcA 和 rtcB 的表达，也与 sigma(54) 的参与一致，rtcA/rtcB mRNA 转录起始位点上游区域包含 -12 和 -24 元件，分别是 TTGCA 和 TGGCA，这是sigma(54) 依赖性启动子。敲除实验证明环化酶基因不是必需的，讨论了环化酶在 RNA 代谢中的可能功能。
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 (K-m = 20 mu M), whereas GTP (K-m = 100 mu M) and other nucleoside triphosphates (NTPs) act less efficiently, The enzyme undergoes nucleotidylation in the presence of [alpha-P-32]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 sigma(54)-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 sigma(54)-dependent manner, Also consistent with the involvement of sigma(54) 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 sigma(54)-dependent promoters. The cyclase gene is nonessential as demonstrated by knockout experiments, Possible functions of the cyclase in RNA metabolism are discussed.