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

摘要

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, steadystate kinetic studies were conducted with chemicaly synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligorionucleotide 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 when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k_(cat)s similar to that for the normal substrate (full-length tRNA~(Phe) unmodified at A37), although the K_m for minihelix~(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely ocnserved 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 k_cat compared to minihelix~(Phe) and a 13-fold increase 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 in which the loop sequence of E. coli tRNA~Phe) was preserved, but the 5 base pair helix stem was completely disurpted 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.