Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.
Escherichia coli 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 for 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 RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.
Pel
H J
HJ
Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand.
Moffat
J G
JG
Ito
K
K
Nakamura
Y
Y
Tate
W P
WP
D019204:GTP-Binding Proteins / Q000737:chemistry; D006147:Guanine / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D010454:Peptide Termination Factors; C041460:peptide chain termination release factor 2; C087971:peptide-chain-release factor 3; D006153:Guanosine Diphosphate; D006147:Guanine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D019204:GTP-Binding Proteins