In vitro recapitulation of the evolution of tRNA nucleotidyltransferases (CCA-adding enzymes)

作为将核酸遗传密码翻译成蛋白质的接头分子，tRNA分子在细胞中起着至关重要的作用。为了装载同源氨基酸，每个tRNA在3 '末端携带不变的CCA三联体。该序列由CCA添加酶（tRNA核苷酸转移酶）构建和维持，该酶在迄今为止分析的每种生物体中发现。有趣的是，在一些生物体中，这种活性由两种不同的蛋白质组成：在这里，CCA添加是由同源CC-和A-添加酶之间的协作催化的。这些核苷酸转移酶密切相关，与细菌CCA添加酶和poly（A）聚合酶一起属于聚合酶β超家族的II类。CCA-，CC-和A-添加酶的高度序列相似性表明，这些不同的活动不仅有一个共同的祖先，但甚至在进化过程中相互转换。使用与体内选择系统相结合的体外进化策略，我们希望（1）概括CC-和A-添加酶进化为完整的CCA酶。因此，我们将（2）确定单个酶结构域的进化变化，这些变化是获得预期功能所必需的。结合最近发表的tRNA核苷酸转移酶的晶体结构，这些结果将有助于（3）深入了解这些酶的底物识别和催化机制。

Being the adapter molecules that translate the genetic code of nucleic acid into protein, TRNA molecules play an essential role in the cell. In order to be charged with the cognate amino acid, each TRNA carries die invariant CCA-triplet at the 3'-temiinus. This sequence is constructed and maintained by the CCA-adding enzyme (TRNA nucleotidyltransferase) that is found in each organism analyzed so far. Interestingly, in some organisms this activity is composed of two distinct proteins: here, CCA addition is catalyzed by a collaboration between homologous CC- and A-adding enzymes. These nucleotidyltransferases are closely related and belong - together with bacterial CCA-adding enzymes and poly(A) polymerases - to the class II of the polymerase beta superfamily. The high sequence similarity of CCA-, CC-, and A-adding enzymes suggests that these distinct activities not only have a common ancestor, but even interconverted during evolution. Using an in vitro evolution strategy combined with an in vivo selection system, we want to (1) recapitulate the evolution of CC- and A-adding enzymes into a complete CCA enzyme. Thereby we will (2) identify the evolutionary changes in individual enzyme domains which are required for the intended gain of function. Together with the recently published crystal structures of TRNA nucleotidyltransferases, the results will (3) give insights into the mechanism of substrate recognition and catalysis of these enzymes.