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 incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg(2+) and 37 degrees C. Filter binding assays conducted in the absence of magnesium 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 microM 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 decrease 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 ribosome-bound cascade yellow-Met-tRNA(f) and eosin-Met-tRNA(f) were as puromycin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides.