In vitro recapitulation of the evolution of tRNA nucleotidyltransferases (CCA-adding enzymes)
Zusammenfassung der Projektergebnisse
The application of an in vivo screening system in E. coli to identify features responsible for the restricted catalytic activities of CC-adding enzymes turned out to be not feasible. The reasons for this are twofold. First, focusing on the gain of A-incorporation in CC-adding enzymes is not possible as there are several endogenous A-adding activities that can substitute for this incorporation, leading to false positive candidates. Second, our alternative rational approach showed that CC-adding enzymes evolved by the deletion of an essential lever element that controls the specificity switch from CTP to ATP incorporation. A screening system based on point mutations would not allow identifying such deletions. However, with this knowledge, we were able to modify the screening system for the search for alternative templating amino acids in CCA-adding enzymes. Currently, we are characterizing the first obtained candidates in vitro. Yet, we consider our project as very successful, as we could identify the nature of the catalytic restriction in CC-adding enzymes, clarifying thereby the evolution of these enzymes. Furthermore, we could describe the function of a lever element in CCA-adding enzymes that positions the templating amino acids in the nucleotide binding pocket, leading to a change in nucleotide specificity. Surprisingly, this important element displays a very unusual evolutionary behavior. While all other parts of the catalytic core show a concerted evolution, resulting in conserved sequence motifs, the lever element shows no conservation between the investigated CCA-adding enzymes. Obviously, it evolves independently of other parts of the catalytic core, but in context with the rest of the individual enzyme, leading to the appearance of genus-specific consensus sequences in this element. Taken together, we think that our results are a considerable contribution to the understanding of function as well as evolution of this fascinating type of nucleotidyltransferases.
