转移RNA（tRNA）在其3′端有一个CCA序列，该序列在tRNA基因中没有被编码。在多数生物中，CCA-添加酶将核苷转移给未成熟的tRNA。CCA-添加酶是一种独立于模板的RNA聚合酶，它竟然“知道”要添加哪些核苷，“知道”只添加三个，“知道”只添加到tRNA。科学家提出各种不同模型来解释这种神奇的酶是怎样发挥作用的，其中第一个模型是30多年前提出来的，但没有一个模型能够给出一幅完整画面。现在，基于核苷结合过程中三个阶段酶-基质复合物的晶体结构，Xiong和 Steitz得以推断出Archaeoglobus fulgidus酶在无寡核苷酸模板时添加CCA的机制。在另一项研究中，Tomita等人确定了来自Aquifex aeolicus的这种酶的晶体结构，它与缺少终端腺苷和一个前来结合的ATP类似物的primer tRNA相结合。两个小组都发现，一个“蛋白模板”摹仿传统聚合酶使用的核苷模板。
Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template
Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.
Figure 1 Overall structures of the AfCCA substrate complexes. a, The AfCCA–tRNA dimer. The tRNA backbone is highlighted with yellow and green coils, whereas AfCCA subunits are represented by red and blue ribbons. b, View of the AfCCA–tRNA dimer rotated by 90° about a horizontal axis from that in a. AfCCA is shown in a surface representation, with blue for positive and red for negative electrostatic potentials. c, The sequences of the AC74 and the ACC75 RNA constructs. The boxed regions indicate sequences identical to those of the acceptor stem of yeast tRNAphe. d, The structure of AfCCA in complex with ACC75. The RNA is in a stick representation whereas the enzyme is in a ribbon representation with head, neck, body and tail domains coloured magenta, green, blue and cyan, respectively. e, Superposition of the complexes with tRNA in yellow and the ACC75 duplex in white.
Figure 2 The progressive refolding of the 3' terminus. The nucleotides A73, C74, C75 and A76 are coloured yellow, blue, orange and red, respectively. Selected protein side chains are shown with hydrogen-bonding interactions represented by dashed lines. a, The AC74 complex with the incoming CTP (orange). The cyan sphere represents metal ion B and the green sphere represents metal ion A, which is observed in a different Mn2+ ion-soaked crystal. b, The ACC75 complex with the incoming ATP (red). c, The tRNA complex.
Figure 3 The two-metal-ion geometry and orientations of primer duplexes in the two classes of CCA-adding enzyme and in Pol . a, Superimposition of the active site of the AC74 complex and that of Pol . AfCCA is colour-coded as in Fig. 1d with the catalytic carboxylates shown. The AC74 duplex RNA is shown in brown with the primer terminal C74 and the incoming CTP in full atom colouring. Pol DNA and the incoming dCTP are in light blue. The anomalous difference electron densities (3 ) arising from the Mn2+ ions (cyan) are shown in green. b, Different orientations of the primer duplexes in class I (red) and class II (yellow) CCA-adding enzymes, and in Pol (blue). The surface representation of the class II CCA-adding enzyme is shown with the modelled tRNA acceptor stem and TC stem loop10. The primer duplexes in the structures of AfCCA and Pol ternary complexes are aligned onto the class II BstCCA enzyme by superimposing their homologous head domains (magenta).
Figure 4 Nucleotide specificity. a, b, Hydrogen-bonding complementarity between the base of the incoming NTP and the nucleotide-binding site. The incoming NTPs and selected protein side chains are in full atom colouring with hydrogen-bonding interactions represented by dashed lines. The nucleotides C74 and C75 are shown in blue and orange, respectively, whereas other nucleotides are in grey. The cyan sphere represents metal ion B. c–f, Changes in the size of the nucleotide-binding pocket at the last two stages of nucleotide addition. The enzyme is shown in a semi-transparent surface representation with selected side chains shown underneath. The colouring of the nucleotides is the same as in Fig. 2. c, The AC74 complex with the observed incoming CTP (orange). d, The ACC75 complex with observed incoming ATP (red). e, The AC74 complex with an incorrect incoming ATP (blue) modelled by superimposing its triphosphate on that of the observed CTP in c. f, The ACC75 complex with an incorrect incoming CTP (blue) modelled by superimposing its triphosphate on that of the observed ATP in d.
Figure 5 Movement of the head domain. AfCCA enzymes from the AC74 (blue), the ACC75 (red) and the tRNA (green) complexes are aligned through superimposing their tRNA-binding domains. The ACC75 RNA is shown in grey. Arg 224 and Asp 61, which are residues situated at opposite sides of the nucleotide-binding pocket, are shown.
