Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity

Abstract

Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.

Adaptation of theRomanomermis culicivoraxCCA-adding enzyme to miniaturized armless tRNA substrates

“…Together with the corresponding enzymes from E. coli (EcoCCA; an organism exclusively carrying conventional cloverleaf-like tRNAs) and H. sapiens (HsaCCA; an organism carrying conventional cytosolic as well as moderately reduced mt-tRNAs), the purified enzyme was tested in vitro for activity. As substrates, three different radioactively labeled tRNA transcripts were generated by in vitro transcription (Figure 2A), as it is well established that tRNA nucleotidyltransferases from all kingdoms readily accept in vitro transcripts lacking base modifications (Okabe et al, 2003;Hoffmeier et al, 2010;Ernst et al, 2018;Erber et al, 2020).…”

“…tRNA Phe from Saccharomyces cerevisiae is one of the best characterized tRNAs and represents a standard substrate for many tRNA-interacting enzymes (Oommen et al, 1992;Loria & Pan, 2000;Ernst et al, 2018), since the unmodified in vitro transcript folds into a structure almost identical to the native tRNA (Shi & Moore, 2000;Byrne et al, 2010).…”

“…To investigate the substrate preferences of HsaCCA and RcuCCA in more detail, a Michaelis-Menten kinetics analysis was performed. Due to the limited RNA solubility, excessive saturating amounts of tRNA cannot be used, and the obtained parameters represent apparent values (Tomita et al, 2004;Tomita et al, 2006;Hoffmeier et al, 2010;Wende et al, 2015;Ernst et al, 2018). As EcoCCA showed no activity on armless tRNAs, this enzyme was excluded from further analysis.…”

“…To identify the contribution of individual enzyme regions to the recognition of armless tRNAs as substrates for CCA-addition, we followed a strategy that we successfully applied to investigate several CCA-adding enzymes concerning their enzymatic features (Betat et al, 2004;Tretbar et al, 2011;Ernst et al, 2018). We reciprocally exchanged N-and C-termini of While these reactions indicate that both enzyme parts participate in the adaptation to the armless tRNA substrates, the contribution of the catalytic core seems to have a greater impact on the acceptance of these substrates.…”

“…Nucleotide incorporation assays were performed as mentioned above. 5 pmol of tRNA were incubated with 1, 5, 10, 25 and 50 arbitrary units of enzyme for 30 min at 20°C and analyzed as described (Hoffmeier et al, 2010;Ernst et al, 2018).…”