Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2

The complicity of initiation factor 2 (IF-2) in causing the observed low in corporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) du ring protein synthesis in an in vitro coupled transcription/translation sys tem was investigated. The low incorporation in comparison to formyl-methion ine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or cou marin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg2+ and 37 degrees C. Filter binding assays conducted in the absence of ma gnesium ions on fMet-tRNA(f) eosin-Met-tRNA(f) and cascade yellow-Met-tRNA( f) confirmed the previously reported value for the dissociation constant of fMet-tRNA(f) of about 1 mu M and placed the binding constants for the two fluorophore derivatives about three-fold higher. Binding of the fluorophore -Met-tRNA(f) species to salt-washed ribosomes showed a more significant dec rease compared to fMet-tRNA(f). Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF-2 was observed in each case. All ribo some-bound cascade yellow-Met-tRNA(f), and eosin-Met-tRNA(f) were as puromy cin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fl uorophore-Met-tRNA species in partial reactions of initiation may account f or the reduced incorporation of these probes at the N terminus of polypepti des. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Edit ions scientifiques et medicales Elsevier SAS.