CCA-adding enzymes with increased substrate affinities: strategies and consequences for adaptation to RNA substrates

tRNA核苷酸转移酶是唯一已知的在其底物（tRNA）上合成特定序列（C-C-A）而不使用外部核酸模板的RNA聚合酶。相反，它们要么依赖于tRNA底物和催化核心中保守的精氨酸残基的组合（I类），要么依赖于氨基酸模板，其中特定的侧链与传入的CTP和ATP形成沃森-克里克样氢键（II类）。后一类这样的CCA添加酶在细菌和真核生物中发现，并显示出非常独特的特征。虽然代表有效的催化剂，但这些酶通常具有非常低的tRNA底物亲和力。我们最近观察到这一规则的几个例外，并确定了II类CCA添加酶表现出增强的tRNA亲和力。在大多数情况下，这些酶必须处理高度奇异的微型发夹状tRNA，增强的底物相互作用似乎代表了一种进化策略，以识别和结合这种非常规底物进行聚合。此外，从Gammaproteobacteria的祖先CCA添加酶的重建也表现出增强的tRNA亲和力。有趣的是，我们的数据表明，所确定的酶遵循不同的策略，这种有效的底物结合。在这里，我们建议调查基板相互作用的各种策略及其对聚合效率和保真度的后果。在生物信息学方法中，我们将进一步确定处理不寻常的tRNA底物的酶候选物，并且这些酶也将在体内和体外详细表征。此外，我们的生物信息学分析揭示了大量不寻常的第二类酶在古细菌（通常只有第一类被发现），我们将研究它们的功能和共同进化与额外存在的古细菌第一类酶。通过这些项目，我们将了解II类CCA添加酶如何以及为什么在底物识别和聚合模式方面表现出如此令人惊讶的进化可塑性，以及I类和II类酶的不寻常组合如何在葡萄球菌中合作。

tRNA nucleotidyltransferases are the only known RNA polymerases that synthesize a specific sequence (C-C-A) on their substrate (tRNA) without using on an external nucleic acid template. Instead, they either rely on a combination of tRNA substrate and a conserved arginine residue in the catalytic core (class I, in Archaea) or on an amino acid template, where specific side chains form Watson-Crick-like hydrogen bonds to the incoming CTP and ATP (class II). The latter class of such CCA-adding enzymes is found in bacteria and eukaryotes and shows a very peculiar feature. While representing efficient catalysts, these enzymes usually have a very low tRNA substrate affinity. We have recently observed several exceptions to this rule and identified class II CCA-adding enzymes exhibiting an enhanced tRNA affinity. In most cases, these enzymes have to deal with highly bizarre miniaturized hairpin-like tRNAs, and the enhanced substrate interaction seems to represent an evolutionary strategy to recognize and bind such unconventional substrates for polymerization. In addition, the reconstruction of an ancestral CCA-adding enzyme from Gammaproteobacteria also showed enhanced tRNA affinity. Interestingly, our data indicate that the identified enzymes follow different strategies for this efficient substrate binding. Here, we propose to investigate the various strategies for substrate interaction and their consequences on polymerization efficiency and fidelity. In a bioinformatic approach, we will identify further enzyme candidates dealing with unusual tRNA substrates, and these enzymes will be also characterized in detail in vivo as well as in vitro. Furthermore, our bioinformatic analysis revealed a large number of unusual class II enzymes in Archaea (where usually only class I is found), and we will investigate their functionality and co-evolution with the additionally present archaeal class I enzymes. With these projects, we will learn how and why class II CCA-adding enzymes show such a surprising evolutionary plasticity in terms of substrate recognition and polymerization mode and how the unusual combination of class I and class II enzymes collaborate in Archaea.