Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis

Nucleic Acids Res. 1999 Sep 15;27(18):3631-7. doi: 10.1093/nar/27.18.3631.

Abstract

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

Publication types

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

MeSH terms

  • Adenosine Monophosphate / metabolism
  • Base Sequence
  • Binding Sites
  • Catalysis
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Fluorescence
  • Kinetics
  • Mutation
  • Nucleic Acid Conformation
  • RNA, Transfer, Amino Acyl / biosynthesis*
  • RNA, Transfer, Amino Acyl / chemistry
  • RNA, Transfer, Amino Acyl / metabolism
  • RNA, Transfer, Gln / chemistry
  • RNA, Transfer, Gln / genetics
  • RNA, Transfer, Gln / metabolism
  • RNA, Transfer, Trp / chemistry
  • RNA, Transfer, Trp / genetics*
  • RNA, Transfer, Trp / metabolism*
  • Substrate Specificity
  • Thermodynamics
  • Tryptophan / metabolism
  • Tryptophan-tRNA Ligase / metabolism*

Substances

  • RNA, Transfer, Amino Acyl
  • RNA, Transfer, Gln
  • RNA, Transfer, Trp
  • Adenosine Monophosphate
  • Tryptophan
  • Tryptophan-tRNA Ligase