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.
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ABSTRACT: In all organisms tRNAs play the essential role of connecting the genetic information found in DNA with the protein synthesis machinery ensuring fidelity during translation. Following transcription tRNAs undergo a number of processing events including numerous post-transcriptional modifications that render a tRNA molecule fully functional. The effects of some modifications go beyond simply affecting tRNA structure and can alter the meaning of the tRNA. This review will summarize the current state of the tRNA editing field, highlighting how editing affects tRNA structure and function in various organisms. It will also discuss recent data that hints at connections between editing and modification that may be exploited by cells to modulate a tRNA's role in translation.Seminars in Cell and Developmental Biology 10/2011; 23(3):269-74. DOI:10.1016/j.semcdb.2011.10.009 · 5.97 Impact Factor
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ABSTRACT: Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.RNA 07/2011; 17(7):1296-306. DOI:10.1261/rna.2748211 · 4.62 Impact Factor
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ABSTRACT: A algorithm is presented to predict the intensity and timing of a single stimulus required to annihilate spontaneously occurring action potentials in the Hodgkin-Huxley model. Elevation of the potassium equilibrium potential causes oscillations in the V, m, h and n parameters. Equations describing the time-varying behavior of these parameters can be used predict the pulse width, coupling interval of a single anodic pulse applied two consecutive action potentials the activity. Results showed that with the estimated parameters was able to completely annihilate the action potentialsEngineering in Medicine and Biology Society, 1997. Proceedings of the 19th Annual International Conference of the IEEE; 02/1997