Pre-steady-state kinetic studies on ribosomal translocation.

Publisher Summary This chapter describes the approach taken in the laboratory for studying the kinetics of partial reactions of translocation using the fluorescence stopped-flow technique. The parallel measurement of both intensity and polarization of fluorescence in many cases provides complementary information. In addition, the polarization values contain valuable structural information. The use of energy transfer-modulated fluorescence as an observable in kinetic measurements offers several advantages as compared to single chromophore experiments. First of all, the amplitudes of the signal changes are larger. Furthermore, the amplitudes, which usually do not yield much information, can in the case of energy transfer changes be interpreted in terms of changes of distances between donor and acceptor fluorophores in the particular reaction steps. Finally, due to the dependence of the energy transfer efficiency on both separation and orientation of the fluorophores, structural transitions, which may not show up in the signals of the individual fluorophores, will in the double-label experiment be reported by energy transfer changes with high probability.