Rate constants for a number of the assembly reactions involved in forming Escherichia coli ribosome initiation complexes have been measured. These reactions were monitored in a stopped-flow device in which Rayleigh scattering and fluorescence anisotropy were followed as a function of time. Fluorescence was induced by laser excitation modulated at 50 kHz. Aminoacyl-tRNA, initiation factor 3 (IF3), and 70S ribosomes were labeled with fluorescent probes. The light-scattering and fluorescence data show that the antiassociation model for IF3 function cannot be correct. IF3 can be considered to act as an effector in an allosteric model for ribosome function. Fluorescence anisotropy stopped-flow experiments provided rate constants for the binding of IF3 to both 30S subunits and to the intact 70S ribosome. Aminoacyl-tRNA's and nucleotide triplets appear to bind rapidly to 70S ribosomes and then a slow first-order conformational change occurs.
ASJC Scopus subject areas