Poster abstracts

Poster number 53 submitted by Brian Smith

Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNAHis guanylyltransferase

Brian A Smith (Department of Biochemistry Ohio State University)

Abstract:
In yeast the tRNAHis guanylyltransferase (yThg1) catalyzes the non-Watson-Crick (WC) dependent incorporation of a single guanosine residue to tRNAHis at the -1 position (G-1) across from the discriminator nucleotide A73 using an unusual 3'-5' nucleotidyl transfer reaction. G-1 serves as a necessary recognition element for histidyl-tRNA synthetase and is conserved among nearly all tRNAHis species. However, it has also been demonstrated that, when presented with a C73 containing tRNAHis variant substrate, yThg1 carries out a second distinct reaction in vitro, adding multiple sequential nucleotides to the polynucleotide chain in a template dependent 3'-5' polymerization reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs)are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity to any other known enzyme family that could be used to suggest the mechanism by which the unusual 3'-5' addition reaction is catalyzed. However, the recently determined high resolution crystal structure of Thg1 reveals remarkable structural similarities between canonical DNA/RNA polymerases and eukaryotic Thg1, suggesting that 3'-5' and 5'-3' nucleotide addition reactions share a common evolutionary origin. We have used transient kinetics to measure the pseudo-first order forward rate constants for the three steps of the G-1 addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (kaden), nucleotidyl transfer (kntrans), and removal of pyrophosphate from the G-1 containing tRNA (kppase). Using this kinetic framework in conjunction with the crystal structure of nucleotide-bound Thg1, we have implicated an essential role for two-metal-ion chemistry in all three chemical steps of the G-1 addition reaction. Furthermore we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, R133) that participate in binding the incoming GTP for nucleotidyl transfer. We also propose that the pyrophosphatase step could act as a determinant for 3'-5' polymerization, as removal of the activated 5' end precludes further nucleotide addition. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNAHis maturation.

Keywords: tRNA, guanylyltransferase