Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases
Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases
复制标题
DOI：
10.1080/15476286.2015.1018502
复制
发表时间：
2015-04-01
期刊：
影响因子：
4.1
通讯作者：
Moerl, Mario
中科院分区：
文献类型：
--
作者：
Ernst, Felix G. M.;Rickert, Christian;Moerl, Mario
CCA 添加酶是高度特异性的 RNA 聚合酶，可合成并维持 tRNA 3 端的 CCA 序列。该核苷酸三联体是 tRNA 被氨酰化并参与蛋白质生物合成的先决条件。在 CCA 添加过程中，这些酶催化核心中的一组高度保守的基序负责准确的顺序核苷酸掺入。在核苷酸结合口袋中，三个氨基酸残基与传入的 CTP 和 ATP 形成类似 Watson-Crick 的碱基对。这些模板氨基酸的重新定位将酶的特异性从 CTP 识别转变为 ATP 识别。然而，这种重要的结构重排背后的机制尚不清楚。在这里，我们发现基序 C（其实际功能尚未确定）有助于聚合过程中核苷酸特异性的转换。人类酶的生化表征以及 EPR 光谱测量表明，突变该基序中高度保守的氨基酸位置 D139 会干扰 AMP 掺入并影响酶中的域间运动。我们提出了一个作用模型，其中基序 C 形成一个灵活的弹簧元件，调节酶头和体结构域的相对方向，以适应 tRNA 不断增长的 3 端。此外，这些构象转变引发模板氨基酸的重新排列，从而在 CCA 合成过程中将核苷酸结合袋的特异性从 CTP 切换为 ATP。
CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.