Martin Georges, Möglich Andreas, Keller Walter, Doublié Sylvie
Department of Cell Biology Biozentrum, University of Basel, Switzerland.
J Mol Biol. 2004 Aug 20;341(4):911-25. doi: 10.1016/j.jmb.2004.06.047.
Polyadenylation of messenger RNA precursors is an essential process in eukaryotes. Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner. Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive. In a recent bovine PAP structure complexed with an analog of ATP and Mn2+, strictly conserved residues interact selectively with the adenine base, but the nucleotide was found in a "non-productive" conformation. Here we report a second bovine crystal structure, obtained in the presence of Mg2+, where 3'-dATP adopts a "productive" conformation similar to that seen in yeast PAP or DNA polymerase beta. Mutational analysis and activity assays with ATP analogs suggest a role in catalysis for one of the two adenine-binding sites revealed by our structural data. The other site might function to prevent futile hydrolysis of ATP. In order to investigate the role of metals in catalysis we performed steady state kinetics experiments under distributive polymerization conditions. These tests suggest a sequential random mechanism in vitro in the presence of ATP and RNA, without preference for a particular order of binding of the two substrates. In vivo, however, where polyadenylation is processive and the primer does not dissociate from the enzyme, an ordered mechanism with the primer as the leading substrate is more likely.
信使核糖核酸前体的聚腺苷酸化是真核生物中的一个重要过程。聚（A）聚合酶（PAP）是核苷酸转移酶家族的成员之一，该家族还包括DNA聚合酶β，它以不依赖模板的方式在mRNA的3'末端掺入ATP。尽管哺乳动物和酵母PAP的结构已为人所知，但其ATP选择机制仍不清楚。在最近一个与ATP类似物和Mn2+复合的牛PAP结构中，严格保守的残基与腺嘌呤碱基选择性相互作用，但发现该核苷酸处于“非生产性”构象。在此我们报告在Mg2+存在下获得的第二个牛晶体结构，其中3'-dATP采用类似于酵母PAP或DNA聚合酶β中所见的“生产性”构象。用ATP类似物进行的突变分析和活性测定表明，我们的结构数据揭示的两个腺嘌呤结合位点之一在催化中起作用。另一个位点可能起到防止ATP无效水解的作用。为了研究金属在催化中的作用，我们在分布聚合条件下进行了稳态动力学实验。这些测试表明，在ATP和RNA存在的情况下，体外存在一种顺序随机机制，对两种底物的结合顺序没有偏好。然而，在体内，聚腺苷酸化是持续进行的，引物不会从酶上解离，更可能是一种以引物作为主要底物的有序机制。
