Dataset Information

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

ABSTRACT: Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.

SUBMITTER: Hodak JH

PROVIDER: S-EPMC1180751 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

Hodak Jose H JH Downey Christopher D CD Fiore Julie L JL Pardi Arthur A Nesbitt David J DJ

Proceedings of the National Academy of Sciences of the United States of America 20050715 30

Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, confor ...[more]

PMID: 16024731

Similar Datasets

Project description:Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.

Dataset Information

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

Publications

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets