Talk abstracts
Talk on Friday 02:15-02:30pm submitted by Grace Johnecheck
Substrate Recognition by Two 3' to 5' RNA Polymerases in Dictyostelium discoideum
Grace Johnecheck (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University), Yicheng Long (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University)
Abstract:
Unlike most polymerases that act in the 5' to 3' direction, tRNAHis guanylyltransferase (Thg1) synthesizes RNA 3' to 5'. Thg1 catalyzes an essential reaction during tRNAHis processing by adding a G nucleotide on the 5' end of the tRNA in most eukaryotes, forming an identity element for tRNA aminoacylation. Recent characterization of Thg1 homologs with alternative specificities, known as Thg1-like proteins (TLPs), raises questions about how distinct substrates are recognized by different members of this highly conserved enzyme family. In the slime mold Dictyostelium discoideum (Ddi), DdiThg1 adds G to the 5' end of cytosolic (cy-) tRNAHis, while DdiTLP2 catalyzes the same reaction, but only with mitochondrial (mt-) tRNAHis substrates. In vivo and in vitro, these two enzymes exhibit strict specificity for their respective tRNA substrates. Moreover, unlike Thg1, DdiTLP2 does not depend on the GUG anticodon for tRNAHis recognition, suggesting different mechanisms for tRNA recognition by these two enzymes. We aimed to determine the molecular basis for the distinct RNA substrate specificities of DdiThg1 and DdiTLP2, and thus to provide insight into the general mechanisms of RNA recognition utilized by distinct 3'-5' RNA polymerases. Electrophoretic mobility shift assays (EMSA) revealed no difference in either enzyme's ability to bind to different tRNAs, requiring additional catalytic factors to explain the selective in vitro activities of DdiThg1 and DdiTLP2. Sequence comparison was used to identify a unique residue in DdiTLP2 (R187) that is different from an absolutely conserved D/E residue at the analogous position in the rest of Thg1/TLP family members, including in DdiThg1. The DdiThg1 D150R variant lost the ability to catalyze G-1 addition with cy-tRNAHis and instead gained the ability to act on DdiTLP2’s mt-tRNAHis substrate. Kinetic and conservative amino acid replacement studies suggest that the DdiThg1 D150 residue controls RNA substrate specificity at the adenylation step of the reaction by providing a checkpoint for correct setup of the active site with incoming GTP to be added. Biochemical studies and computational structure prediction suggest that the role of DdiThg1 D150 is H-bonding with the 3'-OH nucleophile on the incoming GTP, which then positions ATP into the correct location for adenylation.
Keywords: RNA polymerase, tRNAHis, tRNA processing