Biochemical and Structural Studies of A-to-I Editing by tRNA:A34 Deaminases at the Wobble Position of Transfer RNA^,

Abstract

Initial RNA transcription produces several tRNAs (one in prokaryotes and plant chloroplasts and seven or eight in eukaryotes) that contain an adenosine (A) at the wobble position (position 34). However, in all cases, adenosine at position 34 is post-transcriptionally converted to inosine (I), producing mature tRNAs without adenosine at the wobble position. The enzymes responsible for this A-to-I conversion in tRNA are tadA (acting as a homodimer) in prokaryotes and the heterodimeric ADAT2-ADAT3 complex in eukaryotes. The genes encoding these proteins are essential for cell viability, illustrating the biological importance of A-to-I editing at the wobble position of tRNA. In this study, recombinant tadA proteins from Escherichia coli, Agrobacterium tumefaciens, and Aquifex aeolicus, as well as the ADAT2-ADAT3 proteins from Saccharomyces cerevisiae, were overexpressed in E. coli and purified to homogeneity by chromatography. Crystallization of a proteolytically cleaved A. tumefaciens tadA (missing the last eight amino acids at the C-terminus) produced high-quality crystals, and the structure was determined at 1.6 A resolution. In addition, enzymatic assays of the wild-type proteins as well as several mutants were carried out using both the full-length E. coli tRNA(arg2) and the truncated anticodon stem-loop motif as substrates. Our biochemical and structural studies, in combination with sequence and structural comparisons with other deaminases, allow us to propose a model of tadA-tRNA interaction that explains the molecular basis of tRNA recognition by tadA. In particular, a conserved FFxxxR motif at the C-terminus, which is unique to tadA, has been identified, and its critical role in tRNA substrate recognition is proposed. Furthermore, the structural study of prokaryotic tadA presented here also sheds light on tRNA substrate recognition and the possible evolutionary origin of the eukaryotic ADAT2-ADAT3 heterodimer.

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

“…Currently, however, nothing is known about the mode of tRNA binding by any of the eukaryotic tRNA deaminases, so it is not clear if similar sequences found at the C terminus of other ADAT2s may provide a tRNA binding function. The data presented support a previous model in which, through evolution and in order to accommodate many different substrates, a domain has been appended away from the enzyme's active site, providing critical substrate binding functions (Elias and Huang 2005).…”

“…Given the remarkable catalytic flexibility of the TbADAT2/3 (able to perform both A to I and C to U editing in vitro) (Rubio et al 2007), our work has concentrated on utilizing this enzyme to explore its mode of tRNA binding. We have demonstrated that, as predicted by Huang and coworkers, the C-terminal end of ADAT2 contains an essential domain for tRNA binding (Elias and Huang 2005). This domain is formed by a string of positively charged amino acids (arginines and lysines) we termed the KR-domain.…”

“…Most recently, Huang and coworkers suggested that eukaryotic deaminases have evolved the ability to utilize many different substrates by acquiring RNA binding motifs that are distinct from active site residues (Elias and Huang 2005). They also suggested that comparison of the bacterial deaminase sequences to those of the two subunits of the S. cerevisiae deaminase reveal an extension at the C terminus of ADAT2, which may harbor the RNA binding function.…”

“…Huang and coworkers have proposed that, for recognition of seven to eight different substrates, a tRNA binding module has been appended to the eukaryal deaminases away from their active site, which in turn led to accumulation of mutations, resulting in active-site relaxation and concomitant multisubstrate specificity (Elias and Huang 2005). They asserted that likely regions in ADAT2 and ADAT3 that show little sequence conservation with the bacterial RNA deaminases might in fact harbor the appended RNA binding domain.…”

Identification of Amino Acid Residues in APOBEC3G Required for Regulation by Human Immunodeficiency Virus Type 1 Vif and Virion Encapsidation

“…One key difference that emerged was the presence of the additional ␣-helix 3 between ␤-strands 4 and 5 in APOBEC proteins, which is absent from the nucleotide cytidine deaminases. Based on the crystal structures of the human adenosine-to-inosine editing enzyme ADAR2 (29), the tRNA adenosine deaminases from Aquifex aeolicus (15,25) and Staphylococcus aureus (27), Saccharomyces cerevisiae cytosine deaminase (23,24), and APOBEC2 (an orphan APOBEC family member whose crystal structure was reported while this work was being reviewed) (41), it is now believed that ␣-helix 3 of APOBEC protein CDA cores crosses the ␤-sheet, resulting in a parallel positioning of ␤-strands 4 and 5. As a consequence, this helix is located toward the same face of the ␤-sheet as helices ␣1 and ␣2 (Fig.…”