A GTP synthase ribozyme with increased GTP turnover.

Before the invention of encoded protein translation, early stages of life likely relied on catalytic RNAs (ribozymes). To test how such a system could have functioned, researchers have developed ribozymes that could have provided central functions. The central function of self-replication would have required templated RNA polymerization of nucleotides, which is energetically driven in today's life forms by the use of nucleoside 5'-triphosphates (NTPs). We previously showed that ribozymes can catalyze the formation of guanosine 5'-triphosphate (GTP) from guanosine and the prebiotically plausible polyphosphorylation reagent cyclic trimetaphosphate (cTmp) by generating a guanosine triphosphorylation ribozyme (GTR) using an in vitro selection in emulsion. This ribozyme (GTR1) had a catalytic rate enhancement of about 18,000-fold but a turnover of only about 1.7. Here, we improved this ribozyme by emulsion selection from a doped library of GTR1 that was metabolically coupled to a polymerase ribozyme. High-throughput sequencing and biochemical analysis identified the most efficient variant of GTR1 with 19 mutations, which increased the GTP turnover number to ~13. Biochemical analysis of this GTR1e revealed biphasic reaction kinetics with an apparent overall K