Fluorescence temperature jump

W. Bujalowski, D. Porschke

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA.This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons. Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe-tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide.The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe. The binding constants evaluated according to this model (Pheamide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe. Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence, which is not compatible with a corresponding binding process to tRNAGlu. Addition of glutamic acid diamide induces an increase of the tRNAPhe · tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe-tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu. The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine. Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhetRNAGlu dimer. A comparison of the enhanced pairing observed in the presence of Mg2+, which is known to induce a 3'-stack conformation of the anticodon loop, with the reduced pairing affinity observed in the presence of aromatic amino acid amides suggests induction of a 5'-stack conformation by the latter ligands.

Original languageEnglish (US)
Pages (from-to)91-98
Number of pages8
JournalZeitschrift fur Naturforschung - Section C Journal of Biosciences
Volume43
Issue number1-2
DOIs
StatePublished - Jan 1 1988
Externally publishedYes

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Keywords

  • Amino Acid Residues
  • Anticodon Binding
  • Fluorescence Temperature Jump
  • Protein Nucleic Acid Interactions
  • tRNA

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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