Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics
AUTOR(ES)
Uter, Nathan T.
FONTE
National Academy of Sciences
RESUMO
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 kcat. Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNAGln. Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (kchem) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine Kd is significantly higher than the steady-state Km 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 kchem by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on kchem and Kd 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=521953Documentos Relacionados
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