Escherichia coli dimethylallyl diphosphate : tRNA dimethylallyltransferase: Essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (D MAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A 37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodo n position (position 36). By use of purified recombinant enzyme, steady-sta te kinetic studies were conducted with chemically synthesized RNA oligoribo nucleotides to determine the essential elements within the tRNA anticodon s tem-loop structure required for recognition by the enzyme. A 17-base oligor ibonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice , while the same molecule formed a duplex structure with a central loop whe n annealed slowly at higher concentrations. Both the minihelix and duplex s tructures gave k(cat)s similar to that for the normal substrate (full-lengt h tRNA(Phe) unmodified at A37), although the K-m for minihelix(Phe) was app roximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, wh ich is completely conserved in tRNAs modified by the enzyme, was found to b e important for modification. Changing A36 to G in the minihelix resulted i n a 260-fold reduction in k(cat) compared to minihelixPhe and a 13-fold inc rease in K-m. An A38G variant was modified with a 9-fold reduction in k(cat ) and a 5-fold increase in K-m. a random coil 17-base oligoribonucleotide i n 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. Final ly, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme do es not make base-specific contacts important for binding or catalysis in th is region.