Knowledge Bank

Post-transcriptional modification is an important step in the generation of many RNAs. For example tRNAHis requires the addition of a 5'-guanosine (G-1), a reaction that is catalyzed by the tRNAHis guanylyltransferase (Thg1). Thg1 and related enzymes, Thg1-like proteins (TLPs), make up the Thg1/TLP enzyme family which spans all three domains of life and whose family members act as unusual RNA polymerases. While other polymerases act to extend a growing polynucleotide chain in the 5'-3' direction, Thg1/TLP family members act as non-canonical 3'-5' polymerases by adding one or more base-paired nucleotides to the 5'-ends of RNA. Despite their shared chemistry, several features distinguish these two types of enzymes. While TLPs are capable of adding nucleotides to a variety of substrates, Thg1 enzymes selectively recognize a single tRNA (tRNAHis) for G-1 addition. Neither the molecular basis for Thg1’s selectivity nor the basis for the observed differences between Thg1 and TLP functions are fully understood. In addition, while the biological function of Thg1 in tRNAHis maturation is well established, the role(s) of most TLPs remain poorly understood. This project aims to address both of those questions through the study of an essential TLP found in the slime mold Dictyostelium discoideum, DdiTLP4. DdiTLP4 exhibits distinct substrate specificity from Thg1, catalyzing nucleotide addition to non-tRNA substrates such as 5S rRNA and class I noncoding RNA (ncRNA) in vitro, although the biological substrates for the enzyme in vivo remain unknown. Site-directed mutagenesis and enzyme assays with purified proteins have been used to investigate the unique features of DdiTLP4 that allow it to recognize ncRNA, and to assess its activity with model stem-loop substrates, in order to compare its activities to the prototypical Thg1 enzyme. Genetic methods have also been used to determine the effects of depletion of DdiTLP4 on D. discoideum development, to assess the effects of this unusual enzyme in biology.