Talk abstracts

Talk on Monday 10:15-10:30am submitted by Brian Smith

Mechanistic insights into 3’--5' nucleotide addition by eukaryotic tRNAHis guanylyltransferase (Thg1)

Brian Smith (Department of Biochemistry Ohio State University), Jane Jackman (Department of Biochemistry Ohio State University)

Abstract:
tRNAHis guanylyltransferase (Thg1) is a highly conserved enzyme found in all domains of life including higher eukaryotes. Thg1 catalyzes the incorporation of a single guanosine residue to the -1 position (G-1) of tRNAHis, 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. 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. 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. G-1 addition occurs via a complex mechanism involving three steps. In the first step, Thg1 activates the 5' end of tRNAHis by formation of an adenylylated intermediate via a 5'--5' phosphoanhydride bond (App-tRNAHis). Subsequently, Thg1 utilizes the 3'-OH of GTP to attack the intermediate in a nucleotidyltransfer step yielding AMP and resulting in the addition of a single GTP to the 5' end of the tRNA. In the final step Thg1 removes pyrophosphate from the G-1 residue in a pyrophosphatase step yielding mature G-1-containing tRNAHis. A complete understanding of this complex reaction mechanism requires isolation and characterization of each catalytic step individually.We have used transient kinetic to measure the pseudo-first order forward rate constants for each step of this reaction (kaden, kntrans and kppase). Using this kinetic framework in conjunction with data from the newly available crystal structure of nucleotide-bound Thg1, we have also identified conserved residues involved in nucleotide binding and the adenylylation and nucleotidyl transfer steps of the G-1 addition reaction. We have also identified residues which when altered to alanine, cause severe defects in tRNA binding which are localized to a region distant from the putative active site, suggesting the possibility of structural rearrangements induced upon tRNA binding.

References:
Samantha J. Hyde,Brian E. Eckenroth, Brian A. Smith,William A. Eberley,Nicholas H. Heintz, Jane E Jackman, and Sylvie Doublie tRNAHis guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases.2010 Proc. Natl. Acad. Sci. USA, doi: 10.1073/pnas.1010436107.

Keywords: tRNA, nucleotidyl transfer, G-1