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

TRNA核苷酸转移酶是已知的唯一一种在其底物(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(在古生代，第I类)，要么依赖于氨基酸模板，在氨基酸模板中，特定的侧链与进入的CTP和ATP形成类似Watson-Crick的氢键(第II类)。后一类添加CCA的酶在细菌和真核生物中发现，并显示出非常特殊的特征。虽然这些酶代表了有效的催化剂，但它们通常具有非常低的tRNA底物亲和力。我们最近观察到了这一规则的几个例外情况，并鉴定出第二类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怪的小型化发夹状tRNA，而增强的底物相互作用似乎代表了一种识别和结合这些非传统底物进行聚合的进化策略。此外，从伽马变形杆菌中重建的祖先CCA添加酶也显示出增强的tRNA亲和力。有趣的是，我们的数据表明，所鉴定的酶遵循不同的策略进行这种有效的底物结合。在这里，我们建议研究底物相互作用的各种策略及其对聚合效率和保真度的影响。在生物信息学的方法中，我们将确定更多处理不寻常tRNA底物的候选酶，这些酶也将在体内和体外进行详细的表征。此外，我们的生物信息学分析揭示了古生物中大量不寻常的II类酶(通常只发现I类酶)，我们将研究它们的功能及其与另外存在的古生物I类酶的共同进化。通过这些项目，我们将了解如何以及为什么第二类CCA添加酶在底物识别和聚合模式方面显示出如此惊人的进化可塑性，以及第一类和第二类酶的不寻常组合如何在古生代中协作。

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.