Talk abstracts

Talk on Friday 07:10-07:30pm submitted by Frank Ragone

Determinates of Binding and Editing of the Adenosine to Inosine tRNA editing complex from Trypanosoma brucei

Frank Ragone (Microbiology The Ohio State University), Otavio Thiemann (Universidade de So Paulo), Jessica Wohlgamuth-Benedum (Microbiology The Ohio State University), Juan Alfonzo (Microbiology The Ohio State University)

Abstract:
Transfer RNAs (tRNA) containing inosine (I) at the first, or "wobble" position of the anticodon have been observed in two of the three domains of life and when present is essential for cell viability. Inosine formation at the wobble position expands the decoding capabilities of a tRNA, as inosine can base pair with adenosine (A), cytidine (C), or uridine (U), allowing the decoding of multiple codons by a single tRNA. The adenosine deaminases acting on tRNA (ADATs) in bacteria (ADATa) are homodimeric enzymes that recognize only one substrate. In eukarya, the enzyme is a heterodimer composed of two sub-units, ADAT2 and ADAT3 that can recognize seven different tRNAs as substrates. ADAT2 is the catalytically active sub-unit while ADAT3 contains a proposed pseudo-active site but may not be directly responsible for deamination. While the tRNA editing mechanism has been elucidated, the precise residues that provide substrate binding to the enzyme are not known. Here we show the contribution of individual residues in ADAT2 and ADAT3 from Trypanosoma brucei in both the ability to catalyze the deamination reaction and the ability to recognize the tRNA substrate. Mutations to the active site and pseudo active site of the editing complex have catalytic effects although the mutant complex is not deficient in binding tRNA. Deletion of a stretch of charged amino acids at the c-terminus of ADAT2 abolishes both the binding and the deamination activity of the editing complex. Our results provide a first glance of the key residues involved in tRNA binding by this group of editing enzymes.

Keywords: RNA Editing, Deaminase