Poster abstracts

Poster number 55 submitted by Jarrett Wilson

Recognition of RNA duplexes by the tRNAHis guanylyltransferase​

Jarrett Wilson (The Ohio State University), Malithi Jayasinghe (The Ohio State University), BrianSmith (The Ohio State University), Maria Abad (The Ohio State University), Jane E. Jackman (The Ohio State University)

Abstract:
tRNAs are fundamental to the central dogma of biology, as they have a long-established role in protein synthesis. Known as the most highly modified RNA molecule, we lack a complete understanding of the many enzymes that have a role in their post-transcriptional maturation. tRNAHis guanylyltransferase (Thg1) is a part of a large family of enzymes that play an important role in the tRNA maturation process. Thg1 has the unique ability to synthesize RNA in the 3’-5’ direction, catalyzing the non-templated addition of a G-1 nucleotide to the 5’ end of tRNAHis in eukaryotes. Thg1s ability to polymerize in the 3'-5' direction is the opposite canonical RNA polymerases. G-1 is an essential identity element for histidyl-tRNA synthetase (HisRS) to recognize tRNAHis from other tRNAs. Recent studies show that despite its high specificity for its known biological substrate (tRNAHis), Thg1 also binds in vitro to varying RNA duplexes with even higher affinity than for tRNA. This raises questions about the molecular basis for Thg1 recognition of these small RNA duplexes that lack the typical structural features of a tRNA. Previous assays identified highly conserved residues in human Thg1 that reduce tRNA binding, including a catalytically important Glu that exhibits the most significant reduction in affinity for tRNAHis when altered to alanine. In this research, I probe whether yeast and human Thg1 utilize the same essential conserved residue to recognize the RNA duplexes as is used to bind to tRNAHis. Filter binding assays are used to measure RNA duplex binding to WT and mutant versions of yeast and human Thg1, providing important insight into Thg1 RNA recognition properties that are currently unknown.​

References:
"Mechanistic insights into a reverse polymerase",Brian Smith, PhD Thesis​
"Exploration of broader substrate specificity, applications, and mechanism of tRNAHis guanylyltransferase-like proteins (TLPs)",Malithi Jayasinghe, PhD Thesis​
Jane E. Jackman and Eric M. Phizicky 2008​
Maria Abad et al. 2011

Keywords: Thg1, tRNAHis