In vitro recapitulation of the evolution of tRNA nucleotidyltransferases (CCA-adding enzymes)

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Being the adapter molecules that translate the genetic code of nucleic acid into protein, TRNA molecules play an essential role in the cell. In order to be charged with the cognate amino acid, each TRNA carries die invariant CCA-triplet at the 3'-temiinus. This sequence is constructed and maintained by the CCA-adding enzyme (TRNA nucleotidyltransferase) that is found in each organism analyzed so far. Interestingly, in some organisms this activity is composed of two distinct proteins: here, CCA addition is catalyzed by a collaboration between homologous CC- and A-adding enzymes. These nucleotidyltransferases are closely related and belong - together with bacterial CCA-adding enzymes and poly(A) polymerases - to the class II of the polymerase beta superfamily. The high sequence similarity of CCA-, CC-, and A-adding enzymes suggests that these distinct activities not only have a common ancestor, but even interconverted during evolution. Using an in vitro evolution strategy combined with an in vivo selection system, we want to (1) recapitulate the evolution of CC- and A-adding enzymes into a complete CCA enzyme. Thereby we will (2) identify the evolutionary changes in individual enzyme domains which are required for the intended gain of function. Together with the recently published crystal structures of TRNA nucleotidyltransferases, the results will (3) give insights into the mechanism of substrate recognition and catalysis of these enzymes.