Poster abstracts

Poster number 75 submitted by Jessica Spears

Specific contributions of ADAT2 and ADAT3 to substrate binding and deaminase activity

Jessica L. Spears (Microbiology, The Ohio State University), Frank Ragone (Microbiology, The Ohio State University), Nina Papavasiliou (Rockefeller University), Jessica Wohlgamuth-Benedum (Microbiology, The Ohio State University), Cecil J. Howard (Microbiology, The Ohio State University)

Abstract:
RNAs from all three domains of life—eukarya, bacteria, and archaea—can act as substrates for numerous posttranscriptional modifications, including tRNA editing. Of particular interest is the deamination of adenosine to inosine that occurs at the first or “wobble” position of the anticodon in tRNAs. Given the ability of inosine to base pair with cytosine, uridine and adenosine, this editing event allows a single tRNA to decode multiple codons. In eukaryotes, adenosine deaminases acting on tRNA (ADATs) are heterodimeric enzymes responsible for conversion of adenosine to inosine. Of the two subunits, ADAT2 is known to be catalytic while the contributions of ADAT3 to catalysis, substrate recognition and binding are unknown. Here we show, through systematic mutations of the ADAT2/3 enzyme from Trypanosoma brucei, the role that both the ADAT2 and ADAT3 subunits play on editing activity. Single amino acid substitutions to the proposed active and "pseudo active" sites of these subunits, show that as expected ADAT2 plays a prominent role in catalysis, but importantly, suggests that ADAT3 also plays a catalytic role. We also show that deletion of a run of lysine and arginine residues at the c-terminus of ADAT2 impairs tRNA binding, defining a potential tRNA binding site on the enzyme that is distant from the active site. This observation provides a first clue as to the basis for substrate recognition for this group of eukaryotic enzymes and has implications for the evolution of new substrate specificities among eukaryotic deaminases.

Keywords: tRNA editing, Adenosine deaminase