Inosine, a guanosine analog, has been known to function in transfer RNAs (tRNAs) for decades. When inosine occurs at the wobble position of the tRNA, it functionally expands the decoding capability of a single tRNA because inosine can base pair with cytosine, adenosine, and uridine. Because inosine is not genomically encoded, essential enzyme(s) are responsible for deaminating adenosine to inosine by a conserved zinc-mediated hydrolytic deamination mechanism. Collectively called ADATs (Adenosine deaminase acting on tRNA), these enzymes are heterodimeric in eukaryotes and are comprised of subunits called ADAT2 and ADAT3. ADAT2 is presumed to be the catalytic subunit while ADAT3 is thought to be just a structural component. Although these enzymes are essential for cell viability and their products (inosine-containing tRNAs) have a direct effect on translation, little is known about ADAT2/3. Questions such as what is ADAT3’s role in enzyme activity, how many zinc ions are coordinated, how many tRNAs are bound per heterodimer per catalytic cycle and what is the nature of the tRNA binding domain were all open questions until this work.
The focus of chapter two is on the specific contributions of each subunit to catalysis. Steady state kinetic measurements with a series of ADAT2/3 mutants show that ADAT3 contributes directly to catalysis via participation in inter-subunit zinc coordination. A molecular model is presented that is corroborated by ICP (inductively coupled plasma) studies which further show that unlike other multimeric deaminase, one zinc ion is necessary and sufficient for deaminase activity. This effectively means that ADAT2/3 has one complete active site and a pseudo-active site (which is not complete because of a naturally missing catalytic glutamate). Furthermore, electrophoresis mobility shift assays (EMSAs) show a predicted 1:1 stoichiometry of tRNA: ADAT2/3 heterodimer.
In chapter three, the focus shifts from the catalytic site and moves to the tRNA binding domain(s). In silico work predicted an RNA binding domain away from the active site and at the C-terminus of ADAT2 (KR-domain). A combination of enzyme kinetics and EMSAs show that not only are the positively charged arginines and lysines critical for substrate binding but also the pseudo-active site is critical for binding. Moreover, these two binding sites work cooperatively to bind and position a single tRNA for catalysis.
The final study presented in chapter four examines inosine formation away from the anticodon. In archaeal tRNAs, A57 in the TΨC loop is first methylated (m1A57) by the SAM-dependent TrmI and then deaminated (m1I57) by an unknown enzyme(s). Using a combination of bioinformatics and protein purification via column chromatography, progress was made towards the final goal of identifying the enzyme responsible for m1A to m1I activity.
In summary this dissertation presents interesting findings with respect to tRNA editing deaminases and fills important data gaps including new idea about the active site works and how protein binds its tRNA substrate.