RRM
2009 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
tRNAHis guanylyltransferase (Thg1) is an essential enzyme in yeast that adds a single G residue at the -1 position (G-1) of tRNAHis, causing extension of the tRNA in the 3'-5' direction. G-1 is a necessary recognition element for HisRS and is conserved among nearly all tRNAHis species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown. Thg1-catalyzed G-1 addition occurs via a complex mechanism involving three steps: adenylylation, nucleotidyltransfer, and removal of the 5' pyrophosphate. A complete understanding of this complex reaction mechanism requires isolation and characterization of each of these catalytic steps individually. Our investigations reveal three intriguing observations. First, using transient kinetic assays we have shown that the first order rate constants for nucleotidyltransfer (kntrans) and removal of pyrophosphate (kppase) increase with increasing pH (6.0-7.5) while the rate constant for adenylylation (kaden) decreases, implying different ionization event(s) for different steps of the Thg1 reaction, and suggesting the likelihood of identifying residues that function independently for each step. Second, despite the fact that there is no obligate role for NTPs as substrates for the pyrophosphatase step, the presence of GTP increases kppase significantly suggesting previously unsuspected role(s) for nucleotides in this reaction. Third, in the presence of Mn+2 at pH > 8.0 we have detected evidence indicative of an adenylylated enzyme intermediate not previously observed. Adenylylated and guanylylated enzyme intermediates are a common feature in the mechanism of ligase and mRNA capping enzymes which also activate polynucleotide substrates via a 5'-5' phosphoanhydride bond.
Keywords: tRNAHis, guanylyltransferase, nucleotidyltransfer
