Characterization of human and bacterial tRNA-specific adenosine deaminases

Many modified nucleotides are known to be present in tRNAs which have different functions and effects in translation and other cellular processes. Inosine at the wobble position (position 34) in tRNAs has an important role in translation because it increases the base pairing possibilities between mRNA and tRNA. In bacteria only tRNAArg2 contains I34. The deamination at this position is catalyzed by tadA (tRNA-specific adenosine deaminase A) which is the first and so far only bacterial deaminase that acts on RNA. This study presents evidence that bacteria encode only one polypeptide that is sufficient for tRNA:adenosine 34 deaminase activity. tadA is encoded by an essential gene thus underscoring the important role of I34 also in bacteria. tadA selectively binds to tRNAArg2, whereas eukaroytic tRNAs are not bound or deaminated by tadA, except when they contain the anticodon loop of tRNAArg. A minisubstrate consisting of the anticodon arm of tRNAArg2 is a substrate for tadA, providing further evidence that important recognition elements are located in this region of the tRNA. Mutational analysis has shown that the sequence UAGC at positions 33 to 36 and a stem-loop structure are sufficient for inosine formation. Thus, the anticodon is the major determinant for tadA acitivity. However, inosine formation is less efficient with the minimal minisubstrate compared to wild-type minisubstrate, suggesting that additional sequences or structures might be required for efficiency. Recombinant human Tad2 and human Tad3 are sufficient to reconstitute tRNA:adenosine 34 deaminase activity. The complex deaminates eukaryotic tRNAs from humans, B. mori and S. cerevisiae and also tRNAArg2 from E. coli. a-hTad2 antibodies deplete the tRNA:adenosine 34 activity from HeLa cell extracts by depleting both subunits, indicating that they form a stable complex. This is further supported by the result that recombinant hTad2 localizes to the nucleus and transports hTad3 into the nucleus. hTad2 and hTad3 cannot be exchanged in vitro with subunits from other organisms, however this is not due to lack of interaction since hTad2 and hTad3 interact with Tad2 and Tad3 proteins from S. cerevisiae and E. coli. Interestingly, the Arg2 minisubstrate is not deaminated by the human tRNA:adenosine 34 deaminase although this enzyme is known to modify full-length tRNAArg2. This result indicates that tRNA binding is different for prokaryotic and eukaryotic Tad2 and Tad3 proteins. The analysis of bacterial and human tRNA:adenosine 34 deaminases extended our knowledge on how these enzymes work and how they are evolutionarily related.