Poster abstracts

Poster number 18 submitted by Grace Johnecheck

Substrate Recognition by Two 3′ to 5′ RNA Polymerases in Dictyostelium Discoideum

Grace Johnecheck (OSBP), Yicheng Long (OSBP), Jane Jackman (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Unlike most polymerases, which act in the 5′ to 3′ direction, tRNAHis guanylyltransferase (Thg1) acts from 3′ to 5′. It adds a G nucleotide on the 5′ end of tRNAHis in most eukaryotes, forming an identity element for aminoacylation by histidyl-tRNA synthetase (HisRS). The recent characterization of several homologs of Thg1, known as Thg1-like proteins (TLPs), raises important questions about general features of substrate recognition by this highly conserved and essential enzyme family. In the slime mold Dictyostelium discoideum (Ddi), DdiThg1 adds a G to the 5′ end of cytosolic (cy-) tRNAHis, while DdiTLP2 catalyzes the same reaction with mitochondrial (mt-) tRNAHis. When tested in vitro, these two enzymes are specific for their respective tRNA substrates. Unlike Thg1, DdiTLP2 does not depend on the GUG anticodon for tRNAHis recognition, suggesting different mechanisms for tRNA substrate recognition in these two enzymes. We investigated the molecular basis for the unique RNA specificity of each enzyme. Using electrophoretic mobility shift assays (EMSA), we measured overall binding affinity of purified DdiThg1 and DdiTLP2 for each tRNA, and revealed no difference in either enzyme's ability to bind to substrate vs. non-substrate tRNA. However, DdiTLP2 formed a complex with Ddi mitochondrial tRNAHis that was not present with Ddi cytosolic tRNAHis, as evident from the presence of a unique band on the EMSA. Based on the appearance of this band only with substrate, but not non-substrate RNA, we hypothesize that this complex might be associated with productive catalysis. Chemical (CMCT) footprinting assays determined that both DdiThg1 and DdiTLP2 alter the chemical reactivity of the D-loop and anticodon loop in similar ways upon binding to tRNA. Understanding of basic features of recognition in 3′ to 5′ polymerases may be exploited for the development of biochemical tools that use the unique activity to modify the 5′ end of target RNA.

Keywords: RNA polymerase, Thg1, TLP