ESCHERICHIA-COLI RELEASE FACTOR-3 - RESOLVING THE PARADOX OF A TYPICAL G-PROTEIN STRUCTURE AND ATYPICAL FUNCTION WITH GUANINE-NUCLEOTIDES

Escherichia cell release factor 3 (RF3) Is a G protein involved In the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically tor a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 In vitro. Using a direct ribosome binding assay, we found that RF3-GDPCP, a GTP analogue form of RF3, ha s a 10-fold higher affinity for ribosomes than the GDP form of the pro tein, and that RF3-GDPCP binds to the ribosome efficiently In the abse nce of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest t hat the paradoxical Inhibitory effect of GTP on RF3 activity In vitro Is most likely due to untimely and unproductive ribosome mediated CTP hydrolysis. Nucleotide-tree RF3 has an intermediate activity and Its b inding to the ribosome exhibits positive cooperativity with RF2. This cooperativity Is absent, however, In the presence of GDPCP. The observ ed activities of nucleotide-free RF3 suggest that It mimics a transiti on state of RF3 In which the protein interacts with the decoding relea se factor while It enhances the efficiency of the termination reaction .