Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.
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.
Ibba
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Department of Molecular Biophysics and Biochemistry Yale University, New Haven, CT 06520-8114, USA. mibba@imbg.ku.dk
Sever
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Praetorius-Ibba
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Söll
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D000249:Adenosine Monophosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005453:Fluorescence; D007700:Kinetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism*
D012346:RNA, Transfer, Amino Acyl; D012351:RNA, Transfer, Gln; D012364:RNA, Transfer, Trp; D000249:Adenosine Monophosphate; D014364:Tryptophan; D014369:Tryptophan-tRNA Ligase