RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The yeast tRNAHis guanylyltransferase (Thg1) is the only known enzyme that catalyzes addition of nucleotides to a polynucleotide chain in the 3'-5' direction. In S. cerevisiae, Thg1 uses 3'-5' addition to add a single essential G residue (G-1) to the 5' end of tRNAHis. Since Thg1 is an essential enzyme that catalyzes a unique enzymatic activity, the Thg1 catalytic mechanism is of great interest. We have constructed a set of Thg1 variants in which highly conserved residues were altered individually to alanine, and analyzed the effects of these alterations on Thg1 activity, using both in vitro and in vivo assays (Jackman & Phizicky, 2008). We identified 12 residues that play significant roles in G-1 addition to tRNAHis in yeast, and a single residue that dramatically affects tRNA substrate selectivity of the enzyme. Transient kinetic approaches are being used to dissect the Thg1 reaction mechanism and to identify specific roles for each residue in catalysis.
Yeast Thg1 also catalyzes a second 3'-5' nucleotide addition reaction, templated 3'-5' polymerization of multiple nucleotides (Jackman & Phizicky, 2006). The universal requirement for G-1 for tRNAHis aminoacylation, and the occurrence of Thg1 homologs in all sequenced eukaryotes, suggests that G-1 addition to tRNAHis is a conserved role of Thg1 in eukaryotes. In contrast, the physiological function of 3'-5' polymerization is not known, in yeast or in any organism. However, Thg1-related cell-cycle defects are observed in both yeast and human cells that are not obviously related to a role for the enzyme in processing tRNAHis, and moreover, Thg1 homologs have been identified in several archaealand bacteria that do not need enzymatic G-1 addition to tRNAHis. To determine whether there are alternative roles for Thg1, possibly related to its ability to catalyze 3'-5' polymerization, we have compared the 3'-5' nucleotide addition activities of Thg1 and several of its homologs. We have observed characteristic differences in catalytic activity of Thg1 from different domains of life that can be exploited to further probe the role of Thg1 in yeast and other organisms.
References:
Jackman JE, Phizicky EM. 2006. tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. Proc Natl Acad Sci U S A 103:8640-8645.
Jackman JE, Phizicky EM. 2008. Identification of critical residues for G-1 addition and substrate recognition by tRNA(His) guanylyltransferase. Biochemistry 47:4817-4825.
Keywords: tRNA, Thg1, enzyme mechanism
