Escherichia coli Dimethylallyl Diphosphate:tRNA Dimethylallyltransferase: Essential Elements for Recognition of tRNA Substrates Within the Anticodon Stem−Loop

Publisher Website
Google Scholar
Abstract
Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP−tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem−loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem−loop of E. coli tRNAPhe formed a stem−loop minihelix (minihelixPhe) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave kcats similar to that for the normal substrate (full-length tRNAPhe unmodified at A37), although the Km for minihelixPhe was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in kcat compared to minihelixPhe and a 13-fold increase in Km. An A38G variant was modified with a 9-fold reduction in kcat and a 5-fold increase in Km. A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNAPhe was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix−loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.