Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Proc Natl Acad Sci U S A. 2004 Oct 5;101(40):14396-401. doi: 10.1073/pnas.0404017101. Epub 2004 Sep 27.

Abstract

Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat). Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln). Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 A distant. By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.

Publication types

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

MeSH terms

  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Anticodon / genetics
  • Anticodon / metabolism
  • Catalytic Domain
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Glutamine / metabolism
  • Kinetics
  • Macromolecular Substances
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • RNA, Bacterial / biosynthesis
  • RNA, Transfer, Amino Acyl / biosynthesis
  • RNA, Transfer, Gln / genetics
  • RNA, Transfer, Gln / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction

Substances

  • Anticodon
  • Macromolecular Substances
  • RNA, Bacterial
  • RNA, Transfer, Amino Acyl
  • RNA, Transfer, Gln
  • Recombinant Proteins
  • Glutamine
  • Amino Acyl-tRNA Synthetases
  • glutaminyl-tRNA synthetase