In order to understand the molecular basis for tRNA:protein recognition by modification enzyme, I studied the kinetics and binding of purified (His6)-tagged Escherichia coli MiaA tRNA prenyltransferase. MiaA uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U. Kinetic studies were performed with several groups of substrates, including the wild-type tRNAPhe and its variants, tRNASer(V) and its variants, tRNASer(II) and one of its variant. I found that the substitution of the base pairs in the region of anticodon stem loop with other base or base pairs resulted in different effects on the modification of tRNA by MiaA. The two most critical elements with respect to i6A37 formation are A36-A37-A38 and the size of anticodon loop. In addition, two other important requirements for i6A37 formation include A-U base pair at position 31-39 and G-C base pair at positions of 30-40, 29-41, and 28-42.
Binding studies were carried with the filter-binding assay and the fluorescence titration assay, and was performed with several substrates, including the synthetic wildtype tRNAPhe, a mutant tRNAPhe(A37G), the anticodon stem-loop of tRNAPhe(ACSLPhe), and ACSLPhe (A11G) which has G at the position 11 instead of A. By using filter-binding assay, I found that the molar ratio of tRNAPhe(wt) bound per MiaA at saturation was 0.5, suggesting that each tRNA molecule at saturation bound to a MiaA dimer, rather than a monomer. In contrast to tRNAPhe(wt) binding, ACSLPhe(wt) bound MiaA in a 1:1 molar ratio at saturation. Fluorescence quenching assay also supported the hypothesis that tRNAPhe(wt) bound to MiaA dimer. The Kdapp was about 0.07 mM for MiaA binding to tRNAPhe (wt) from the protein-excess titrations of filter binding assay. In contrast, the Kdapp was 1.1 mM for MiaA binding to ACSLPhe(wt) from ligand-excess titration of filter binding assay, indicating the Kdapp for ACSLPhe(wt) was 15 fold higher. Consistent with this result, fluorescence titration assay also showed a significant increase in Kd for ACSLPhe(wt) compared to that for tRNAPhe(wt).
Two major conclusions were drawn from the kinetic and binding studies of MiaA. First, the ACSL contains the majority of the recognition set for the i6A37 formation, because changing a base or a base pair in this region decreases or even abolishes the enzyme activity. Second, there are some other elements outside ACSL could partially influence the interaction between MiaA and tRNA, because ACSLPhe(wt) binds much weaker to MiaA than tRNAPhe(wt), as implicated from the Kd values. In addition, ACSLPhe(wt) binds to MiaA monomer, while tRNAPhe(wt) binds to MiaA dimer.
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Kinetic and Binding Properties of the MiaA tRNA Prenyltransferase of Escherichia coli for Mutant tRNAPhe and tRNASer Substrates