Project Details
Projekt
Unusual RNA-polymerases: reaction mechanism and substrate specificity of tRNA nucleotidyltransferases
Subject Area
Biochemistry
Term
from 2012 to 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 219382169
CCA-adding enzymes (tRNA nucleotidyltransferases) represent highly specific RNA polymerases with unusual features. They synthesize the sequence C-C-A at high fidelity at the 3´-end of tRNAs, generating the site of aminoacylation. While they are highly selective for CMP and AMP addition, these enzymes accept all tRNAs within a cell, in eukaryotes even cytosolic and mitochondrial tRNAs that frequently show structural deviations. Besides the acceptor stem, we could identify the discriminator position 73 as an important recognition element. Now we want to clarify the recognition of these elements by CCA-adding enzymes and learn how they influence efficiency and fidelity of the individual steps of CCA-addition. We want to expand our successful ESR analyses and characterize movements of individual domains and structure elements in highly interesting special forms of these enzymes that are required for a specific recognition of tRNA and nucleotides. With the help of specifically incorporated non-natural amino acids that can be cross-linked to neighboring positions, we want to identify intramolecular interacting regions in these enzymes that are essential for functionality.In a detailed phylogenetic study, we have identified highly unusual combinations of different tRNA nucleotidyltransferases in several pro- and eukaryotes. Now we want to characterize these enzymes and analyze their specific functions as well as their substrate spectrum. Furthermore, we want to use bioinformatic ancestral sequence reconstruction to identify common ancestors of such enzymes and to test those as recombinant proteins for substrate specificity, polymerization reaction, temperature optimum and other parameters. With these experiments, we want to clarify whether tRNA nucleotidyltransferases with partial activities (CC- and A-adding enzymes), poly(A) polymerases or other subgroups of CCA-adding enzymes represent the ancestral state in evolution.
DFG Programme
Research Grants
