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

Biochemistry. 2000 May 30;39(21):6546-53. doi: 10.1021/bi992775u.

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 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkyl and Aryl Transferases / chemistry*
  • Alkyl and Aryl Transferases / metabolism*
  • Anticodon / chemistry
  • Anticodon / metabolism
  • Base Sequence
  • Escherichia coli / enzymology*
  • Kinetics
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • Oligoribonucleotides / chemistry
  • Oligoribonucleotides / metabolism*
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / metabolism
  • RNA, Transfer / chemistry*
  • RNA, Transfer / metabolism*
  • Substrate Specificity

Substances

  • Anticodon
  • Oligoribonucleotides
  • RNA, Bacterial
  • RNA, Transfer
  • Alkyl and Aryl Transferases
  • adenylate isopentenyltransferase