Poster abstracts
Poster number 38 submitted by Tracy Roach
Recognition of tRNA substrates by Thg1 family proteins
Tracy M. Roach (The Ohio State University, Department of Chemistry and Biochemistry), Marie-Theres Phler (Institute for Biochemistry, Leipzig University), Heike Betat (Institute for Biochemistry, Leipzig University), Mario Mrl (Institute for Biochemistry, Leipzig University), Jane E. Jackman (The Ohio State University, Department of Chemistry and Biochemistry)
Abstract:
tRNAHis guanylytransferase (Thg1) is responsible for adding G-1 to tRNAHis across from an A73 discriminator nucleotide in eukaryotes. In addition to this 3'-5' nucleotide incorporation at the tRNA 5'-end, addition of CCA to the 3'-end of tRNAHis is also required for histidyl tRNA synthetase to properly recognize and aminoacylate tRNAHis. Both Thg1 and CCA-adding enzyme localize to the cytosol of Saccharomyces cerevisiae, so these essential enzymes could compete for end-matured tRNAHis that is exported after processing in the nucleus. Therefore, we investigated if there is a preferred pathway between 5'- and 3'-end processing of tRNAHis. To evaluate the possibility of a temporal order of processing required in S. cerevisiae, kinetic studies were used to determine rates of nucleotide incorporation by Thg1 and CCA-adding enzyme to various tRNA substrates representing intermediates in this maturation pathway. Our in vitro studies, which based on the observed rates, binding affinities, and effects on incorporation fidelity all support a preferred pathway in which 3'-CCA addition precedes 5'-end maturation in yeast [1].
Based on these data in eukaryotes, we also questioned if a similar tRNAHis processing order is observed in archaeal species that encode Thg1-Like Proteins (TLPs) and CCA-adding enzymes. In Archaea, the analysis is complicated by differences in tRNAHis, which lead TLPs to add multiple nucleotides to the 5'-end base pairing with the 3'-CCA, thus
References:
1. Poehler MT, Roach TM, Betat H, Jackman JE, Moerl M. A temporal order in 5'- and 3'- processing of eukaryotic tRNAHis. Int. J. Mol. Sci. 2019.
Keywords: tRNAHis guanylyltransferase, enzymology, tRNA maturation