Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop.
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 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 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 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 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.
Soderberg
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Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
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D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism*; D000926:Anticodon / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D000926:Anticodon; D009843:Oligoribonucleotides; D012329:RNA, Bacterial; D012343:RNA, Transfer; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase