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

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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.